Interleukin—1 receptor antagonist and uses thereof

ABSTRACT

The present invention provides novel nucleic acids, the novel polypeptide sequences encoded by these nucleic acids and uses thereof. These novel polynucleotide and polypeptide sequences were determined to be a novel Interleukin-1 Receptor Antagonist.

1. RELATED APPLICATIONS

This patent application is a continuation-in-part of U.S. patentapplication Ser. No. 09/523,552 filed Mar. 10, 2000, which is acontinuation-in-part of U.S. patent application Ser. No. 09/457,626filed Dec. 8, 1999, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/417,455 filed Oct. 13, 1999 (issued as U.S. Pat.No. 6,294,655) which is a continuation-in-part of U.S. patentapplication Ser. No. 09/348,942 filed Jul. 7, 1999 (issued as U.S. Pat.No. 6,337,072) which is a continuation-in-part of U.S. patentapplication Ser. No. 09/287,210 filed Apr. 5, 1999 (now abandoned) whichis a continuation-in-part of U.S. patent application Ser. No. 09/251,370filed Feb. 17, 1999 (now abandoned) which is a continuation-in-part ofU.S. patent application Ser. No. 09/127,698, filed Jul. 31, 1998 (nowabandoned), and a continuation-in-part of U.S. patent application Ser.No. 09/229,591 filed Jan. 13, 1999 (now abandoned), which is acontinuation of U.S. patent application Ser. No. 09/099,818 filed Jun.19, 1998 (now abandoned). U.S. patent application Ser. No. 09/127,698and U.S. patent application Ser. No. 09/099,818 are continuations inpart of U.S. patent application Ser. No. 09/082,364, filed May 20, 1998(now abandoned), which is a continuation-in-part of U.S. patentapplication Ser. No. 09/079,909, filed May 15, 1998 (now abandoned),which is a continuation-in-part of U.S. patent application Ser. No.09/055,010, filed Apr. 3, 1998 (now abandoned), all of which areincorporated by reference herein in their entirety.

2. FIELD OF THE INVENTION

The present invention provides novel polynucleotides and proteinsencoded by such polynucleotides, along with therapeutic, diagnostic andresearch utilities for these polynucleotides and proteins.

3. BACKGROUND

Technology aimed at the discovery of protein factors (including e.g.,cytokines, such as lymphokines, interferons, CSFs, chemokines, andinterleukins) has matured rapidly over the past decade. The now routinehybridization cloning and expression. cloning techniques clone novelpolynucleotides “directly” in the sense that they rely on informationdirectly related to the discovered protein (i.e., partial DNA/amino acidsequence of the protein in the case of hybridization cloning; activityof the protein in the case of expression cloning). More recent“indirect” cloning techniques such as signal sequence cloning, whichisolates DNA sequences based on the presence of a now well-recognizedsecretory leader sequence motif, as well as various PCR-based or lowstringency hybridization cloning techniques, have advanced the state ofthe art by making available large numbers of DNA/amino acid sequencesfor proteins that are known to have biological activity by virtue oftheir secreted nature in the case of leader sequence cloning, or byvirtue of the cell or tissue source in the case of PCR-based techniques.It is to these proteins and the polynucleotides encoding them that thepresent invention is directed. In particular, this invention is directedto a novel Interleukin-1 Receptor Antagonist.

Cytokines, such as Interleukin-1, are well known to cause morphologicaland functional alterations in endothelial cells. These alterations occurin part as a result of “endothelial cell activation” Distinctimmune-mediators such as tumor necrosis factor (TNF), interleukin-1(Interleukin-1), and gamma-interferon (IFN) appear to induce differentbut partially overlapping patterns of endothelial cell activationincluding increased procoagulant activity (Bevilaqua (1986) PNAS,83:4533-4537), PGI and 2 production (Rossi (1985), Science,229:174-176), HLA antigen expression (Pober (1987) J. Immunol.,138:3319-3324) and lymphocyte adhesion molecules (Carender (1987) J.Immunol., 138:2149-2154). These cytokines are also reported to causehypotension, vascular hemorrhage, and ischemia (Goldblum et al. 1989,Tracey et al. Science 234:470, 1986). A major dose limiting toxicity ofthese and other biological response modifiers is hypotension andvascular leakage (Dvorak (1989) J.N.C.I., 81 :497-502).

The ability of IL-1 to modify biological responses has been demonstratedin a variety of studies. For example, the administration ofInterleukin-1 to rabbits (Wakabayashi et al., FASEB J 1991;5:338;Okusawa et al. J Clin Invest 1988;81:1162; Ohlsson et al., Nature1990;348:550; Aiura, et al. Cytokine 1991;4:498) and primates (Fischeret al. Am J Physiol 1991;261:R442) has been shown to result inhypotension, tachycardia, lung edema, renal failure, and, eventually,death, depending on the dose. When the serum from the Interleukin-1treated animals is examined, the elevation of other cytokines isevident, mimicking the levels seen in acute pancreatitis in humans.(Guice et al., J Surg Res 1991;51:495-499; Heath et al., Pancreas1993;66:41-45) There is a large body of evidence currently availablewhich supports the role of Interleukin-1 as a major mediator of thesystemic response to diseases such as sepsis and pancreatitis and as anactivator of the remaining members of the cytokine cascade. (Dinarelloet al., Arch Surg 1992;127:1350-1353).

The cytokine Interleukin-1 is a key mediator in the inflammatoryresponse (for reviews, see Dinarello (1991) Blood 77: 1627-1652;Dinarello et al. (1993) New England J. Med. 328:106-113; Dinarello(1994) FASEB J. 8:1314-1325). The importance of Interleukin-1 ininflammation has been demonstrated by the ability of the highly specificInterleukin-1 receptor antagonist protein to relieve inflammatoryconditions (for review, see Dinarello (1991) Blood 77: 1627-1652;Dinarello et al. (1993) New England J. Med. 328:106-113; Dinarello(1994) FASEB J. 8:1314-1325; Dinarello (1993) Immunol. Today14:260-264). Many of the proinflammatory effects of Interleukin-1, suchas the upregulation of cell adhesion molecules on vascular endothelia,are exerted at the level of transcriptional regulation. Thetranscriptional activation by Interleukin-1 of cell adhesion moleculesand other genes involved in the inflammatory response appears to bemediated largely by NF-kappa B (Shirakawa et al. (1989) Molc. Cell Biol.9:2424-2430; Osborn et al., (1989) Proc. Natl. Acad. Sci. USA86:2336-2340; Krasnow et al., (1991) Cytokine 3:372-379; Collins et al.,(1993) Trends Cardiovasc. Med. 3:92-97). In response to Interleukin-1,the NF-kappa B inhibitory factor I kappa B is degraded and NF-kappa B isreleased from its inactive cytoplasmic state to localize within thenucleus where it binds DNA and activates transcription (Liou et al.(1993) Curr. Opin. Cell Biol. 5:477-487; Beg et al., (1993) Mol. Cell.Bid. 13:3301-3310).

Interleukin-1 is also a mediator of septic shock. Septic shock, alife-threatening complication of bacterial infections, affects 150,000to 300,000 patients annually in the United States (Parrillo, J. E.(1989), Septic Shock in Humans: Clinical Evaluation, Pathogenesis, andTherapeutic Approach (2nd ed.) In: Textbook of Critical Care Shoemaker,et al., editors, Saunders Publishing Co., Philadelphia, Pa., pp. 1006).The cardiovascular collapse and multiple metabolic derangementsassociated with septic shock are due largely to bacterial endotoxin(ET), which has been shown to elicit a septic shock-like condition whenadministered to animals (Natanson, et al. (1989), Endotoxin and TumorNecrosis Factor Challenges in Dogs Simulate the Cardiovascular Profileof Human Septic Shock, J. Exp. Med. 169:823). Thus, there is a greatneed for modulators of Interleukin-1.

4. SUMMARY OF THE INVENTION

The compositions of the present invention include novel isolatedpolypeptides, in particular, novel Interleukin-1 Receptor Antagonistproteins (referred to hereafter as IL-1Hy1 or IL-1 Hy1 receptorantagonist), isolated polynucleotides encoding such polypeptides,including recombinant DNA molecules, cloned genes or degenerate variantsthereof, especially naturally occurring variants such as allelicvariants, and antibodies that specifically recognize one or moreepitopes present on such polypeptides.

The compositions of the present invention additionally include vectors,including expression vectors, containing the polynucleotides of theinvention, cells genetically engineered to contain such polynucleotidesand cells genetically engineered to express such polynucleotides.

The isolated polynucleotides of the invention include, but are notlimited to, a polynucleotide encoding a polypeptide comprising the aminoacid sequence of SEQ ID NO: 3 or 5.

The isolated polynucleotides of the invention further include, but arenot limited to, a polynucleotide comprising the nucleotide sequence ofSEQ ID NO: 1, 2, 4, or 6; a polynucleotide comprising the full lengthprotein coding sequence of SEQ ID NO: 1, 2, 4, or 6, and; apolynucleotide comprising the nucleotide sequence of the mature proteincoding sequence of SEQ ID NO: 1, 2, 4, or 6. The polynucleotides of thepresent invention also include, but are not limited to, a polynucleotidethat hybridizes to the complement of the nucleotide sequence of SEQ IDNO: 1, 2, 4, or 6 under stringent hybridization conditions; apolynucleotide which is an allelic variant of any polynucleotide recitedabove; a polynucleotide which encodes a species homolog of any of theproteins recited above; or a polynucleotide that encodes a polypeptidecomprising a specific domain or truncation of the polypeptide of SEQ IDNO: 1, 2, 4, or 6.

The isolated polynucleotides of the invention further include, but arenot limited to a polynucleotide comprising the nucleotide sequence ofthe genomic clone SEQ ID NO: 7 or 8; a polynucleotide assembled from oneor more of the exons of SEQ ID NO: 7 or 8; a polynucleotide assembledfrom one or more of the introns of SEQ ID NO: 7 or 8; a polynucleotideassembled from one or more of the exons of SEQ ID NO: 7 or 8 and one ormore of the introns of SEQ ID NO: 7 or 8; a polynucleotide comprisingthe full length protein coding sequence of SEQ ID NO: 7 or 8; apolynucleotide comprising the nucleotide sequence of the mature proteincoding sequence of SEQ ID NO: 7 or 8.

The polynucleotides of the present invention also include, but are notlimited to, a polynucleotide that hybridizes to the complement of thenucleotide sequence of SEQ ID NO: 7 or 8 under stringent hybridizationconditions; a polynucleotide that hybridizes to the complement of anyone of the introns or exons of SEQ ID NO: 7 or 8 under stringenthybridization conditions; a polynucleotide which is an allelic variantof any polynucleotide recited above; a polynucleotide which encodes aspecies homolog of any of the proteins recited above; or apolynucleotide that encodes a polypeptide comprising a specific domainor truncation of the polypeptide of SEQ ID NO: 7 or 8.

The polynucleotides of the present invention still further include, butare not limited to, a polynucleotide comprising the nucleotide sequenceof the cDNA insert of clone pIL-1Hy273 deposited with the American TypeCulture Collection (ATCC; 10801 University Blvd., Manassas, Va.,20110-2209, U.S.A.); a polynucleotide comprising a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 3 or 5 whichpolynucleotide is assembled from the cDNA insert of clone pIL-1Hy273; apolynucleotide comprising the full length protein coding sequence of SEQID NO: 3 or 5 which polynucleotide is assembled from the cDNA insert ofclone pIL-1Hy273; or, a polynucleotide comprising the nucleotidesequence of the mature protein coding sequence of SEQ ID NO: 3 or 5.

The polynucleotides of the invention additionally include the complementof any of the polynucleotides recited above.

A collection as used in this application can be a collection of only onepolynucleotide. The collection of sequence information or identifyinginformation of each sequence can be provided on a nucleic acid array. Inone embodiment, segments of sequence information are provided on anucleic acid array to detect the polynucleotide that contains thesegment. The array can be designed to detect nucleic acids that areperfectly complementary (full-match) or mismatched to the polynucleotidethat contains the segment. The collection can also be provided in acomputer-readable format.

The isolated polypeptides of the invention include, but are not limitedto, a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or5, a full length protein of SEQ ID NO: 3 or 5; a mature protein codingsequence of SEQ ID NO: 5, preferably having a molecular mass asdetermined by SDS-PAGE of about 16 or 17 kDa, or less, and morepreferably, having a molecular mass by SDS-PAGE of about 15.5 or about16.5 kDa; or a polypeptide encoded by one or more of the exons of SEQ IDNO: 7 or 8.

The polypeptides of the present invention further include, but are notlimited to, a polypeptide comprising the amino acid sequence encoded bythe cDNA insert of clone pIL-1Hy273 deposited with the American TypeCulture Collection (ATCC; 10801 University Blvd., Manassas, Va.,20110-2209, U.S.A.); a full length protein of SEQ ID NO: 3 or 5assembled from the amino acid sequence encoded by the cDNA insert ofclone pIL-1Hy273; or, a mature protein coding sequence of SEQ ID NO: 3or assembled from the amino acid sequence encoded by cDNA insert ofclone pIL-1Hy273, preferably having a molecular mass as determined bySDS-PAGE of about 16 kDa, or less, more preferably, having a molecularmass by SDS-PAGE of about 15.5 kDa.

Protein compositions of the present invention may further comprise anacceptable carrier, such as a hydrophilic, e.g., pharmaceuticallyacceptable, carrier.

The invention also relates to methods for producing a polypeptidecomprising growing a culture of the cells of the invention in a suitableculture medium, and purifying the protein from the culture. Preferredembodiments include those in which the protein produced by such processis a mature form of the protein.

Polynucleotides according to the invention have numerous applications ina variety of techniques known to those skilled in the art of molecularbiology. These techniques include use as hybridization probes, use asoligomers for PCR, use for chromosome and gene mapping, use in therecombinant production of protein, and use in generation of anti-senseDNA or RNA, their chemical analogs and the like. For example, when theexpression of an mRNA is largely restricted to a particular cell ortissue type, polynucleotides of the invention can be used ashybridization probes to detect the presence of the particular cell ortissue mRNA in a sample using, e.g., in situ hybridization.

In other exemplary embodiments, the polynucleotides are used indiagnostics as expressed sequence tags for identifying expressed genesor, as well known in the art and exemplified by Vollrath et al., Science258:52-59 (1992), as expressed sequence tags for physical mapping of thehuman genome.

The polypeptides according to the invention can be used in a variety ofconventional procedures and methods that are currently applied to otherproteins. For example, a polypeptide of the invention can be used togenerate an antibody that specifically binds the polypeptide. Thepolypeptides of the invention can also be used as molecular weightmarkers, and as a food supplement.

Methods are also provided for preventing, treating or ameliorating amedical condition which comprises administering to a mammalian subject atherapeutically effective amount of a composition comprising a proteinof the present invention and a pharmaceutically acceptable carrier.

In particular, the polypeptides and polynucleotides of the invention canbe utilized, for example, as part of methods for the prevention and/ortreatment of disorders involving sepsis, acute pancreatitis, endotoxicshock, cytokine induced shock, rheumatoid arthritis, chronicinflammatory arthritis, pancreatic cell damage from diabetes mellitustype 1, graft versus host disease, inflammatory bowel disease,inflamation associated with pulmonary disease, other autoimmune diseaseor inflammatory disease, allergies or bronchitis (including chronicallergies and chronic bronchitis), an antiproliferative agent such asfor acute or chronic myelogenous leukemia or in the prevention ofpremature labor secondary to intrauterine infections. Treatment ofinflammation resulting from allergic reactions or acute or chronicinfections (caused by viral, bacterial, fungal, protozoan or otherorganisms) is specifically contemplated.

The methods of the present invention further relate to methods fordetecting the presence of the polynucleotides or polypeptides of theinvention in a sample. Such methods can, for example, be utilized aspart of prognostic and diagnostic evaluation of disorders as recitedabove and for the identification of subjects exhibiting a predispositionto such conditions. Furthermore, the invention provides methods forevaluating the efficacy of drugs, and monitoring the progress ofpatients, involved in clinical trials for the treatment of disorders asrecited above.

The invention also provides methods for the identification of compoundsthat modulate the expression of the polynucleotides and/or polypeptidesof the invention. Such methods can be utilized, for example, for theidentification of compounds that can ameliorate symptoms of disorders asrecited above. Such methods can include, but are not limited to, assaysfor identifying compounds and other substances that interact with (e.g.,bind to) the polypeptides of the invention.

The methods of the invention also include methods for the treatment ofdisorders as recited above which may involve the administration of suchcompounds to individuals exhibiting symptoms or tendencies related todisorders as recited above. In addition, the invention encompassesmethods for treating diseases or disorders as recited herein byadministering compounds and other substances that modulate the overallactivity of the target gene products. Compounds and other substances caneffect such modulation either on the level of target gene expression ortarget protein activity.

The invention also specifically provides methods of treating aninflammatory disease state mediated by IL-18 comprising administering toa subject in need thereof an amount of IL-1 Hy1 polynucleotide,polypeptide or agonist of the invention effective to inhibit IL-18activity. Also provided are in vitro and in vivo methods of inhibitingIL-18 activity.

5. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sequence alignment of SEQ ID NO: 3 with human (SEQ IDNO: 30), mouse (SEQ ID NO: 9), rat (SEQ ID NO: 10) and rabbitInterleukin-1 receptor antagonist (SEQ ID NO: 11). A—Alanine;R—Arginine; —Asparagine; D—Aspartic Acid; C—Cysteine; E—Glutamic Acid;Q—Glutamine; G—Glycine; H—Histidine; I—Isoleucine; L—Leucine; K—Lysine;—Methionine; F—Phenylalanine; P—Proline; S—Serine; T—Threonine;W—Tryptophan; Y—Tyrosine; V—Valine; X—any of the twenty amino acids.Gaps are presented as dashes. Amino acid numbers for all four sequencesare labeled accordingly.

FIG. 2 shows the nucleic acid sequences that were obtained from theb²HFLS20W cDNA library using standard PCR, sequencing by hybridizationsignature analysis, and single pass gel sequencing technology. Thesesequences are designated as SEQ ID NOS: 1 and 2. A—adenosine;C—cytosine; G—guanosine; T—thymine; and N—any of the four bases.

FIG. 3 shows the amino acid sequences which correspond to nucleotides 1through 240 of SEQ ID NO: 2. These sequences are designated as SEQ IDNO: 3. A—Alanine; R—Arginine; —Asparagine; D—Aspartic Acid; C—Cysteine;E—Glutamic Acid; Q—Glutamine; G—Glycine; H—Histidine; I—Isoleucine;L—Leucine; K—Lysine; —Methionine; F—Phenylalanine; P—Proline; S—Serine;T—Threonine; W—Tryptophan; Y—Tyrosine; V—Valine; X—any of the twentyamino acids.

FIG. 4 shows the sequence alignment of receptor binding regions of humanInterleukin-1 beta (SEQ ID NO: 12) and human Interleukin-1 receptorantagonist (SEQ ID NO: 13)aligned with a cognate region of SEQ ID NO:3(amino acids 13 through 30 of SEQ ID NO:3). Residues conserved among allthree domains are shown in boldface.

FIG. 5 shows the nucleic acid sequence designated SEQ ID NO: 4(nucleotides 297 through 1282 of SEQ ID NO: 4 correspond to SEQ ID NO:2) and are described in Example 6 . The first eleven nucleic acidsequences correspond to vector sequence.

FIG. 6 shows the amino acid sequence encoded by SEQ ID NO: 4 (aminoacids 76 through 155 of SEQ ID NO: 5 correspond to SEQ ID NO; 3). Thisamino acid sequence is designated SEQ ID NO: 5 and is described inExample 6.

FIG. 7 presents an amino acid alignment of SEQ ID NO: 5 (amino acids 1through 155 of SEQ ID NO: 5) with the cytoplasmic form of human IL-1 Ra( SEQ ID NO: 14; labeled “HUMIL1RASIC”). The homology between these twosequences is discussed in Example 6.

FIG. 8 shows SEQ ID NO: 6 which represents an extension (the underlinedsequence) of the nucleic acid sequence corresponding to SEQ ID NO: 4 andis described in Example 7.

FIGS. 9A-C show the genomic sequence corresponding to SEQ ID NOS: 1, 2,and 4 6. The isolation of the genomic clone (SEQ ID NO: 7 ) from whichthis sequence was derived is described in Example 8. A—adenosine;C—cytosine; G—guanosine; T—thymine. Ambiguous positions are designatedas follows: R indicates A or G; M indicates A or C; W indicates A or T;Y indicates C or T; S indicates C or G; K indicates G or T; V indicatesA or C or G; H indicates A or C or T; D indicates A or G or T; Bindicates C or G or T; and N indicates any of the four bases.

FIGS. 10A-C show a genomic clone (SEQ ID NO: 8) which is an extension ofthe genomic sequence presented in FIGS. 9A-C (SEQ ID NO: 7). SEQ ID NO:8 includes the extended sequence shown in SEQ ID NO: 6 for theInterleukin-1 Receptor Antagonist extension presented in SEQ ID NO: 6.The isolation of this genomic clone (SEQ ID NO: 7 ) from which thissequence was derived is described in Example 11. A—adenosine;C—cytosine; G—guanosine; T—thymine. Ambiguous positions are designatedas follows: R indicates A or G; M indicates A or C; W indicates A or T;Y indicates C or T; S indicates C or G; K indicates G or T; V indicatesA or C or G; H indicates A or C or T; D indicates A or G or T; Bindicates C or G or T; and N indicates any of the four bases.

FIG. 11 shows the dose dependent ability of IL-1Hy1 to inhibit IL-1βinduced PGE₂ production.

6. DETAILED DESCRIPTION 6.1. Definitions

The term “nucleotide sequence” refers to a heteropolymer of nucleotidesor the sequence of these nucleotides. The terms “nucleic acid” and“polynucleotide” are also used interchangeably herein to refer to aheteropolymer of nucleotides. Generally, nucleic acid segments providedby this invention may be assembled from fragments of the genome andshort oligonucleotide linkers, or from a series of oligonucleotides, orfrom individual nucleotides, to provide a synthetic nucleic acid whichis capable of being expressed in a recombinant transcriptional unitcomprising regulatory elements derived from a microbial or viral operon,or a eukaryotic gene.

The terms “oligonucleotide fragment” or a “polynucleotide fragment”,“portion,” or “segment” is a stretch of polypeptide nucleotide residueswhich is long enough to use in polymerase chain reaction (PCR) orvarious hybridization procedures to identify or amplify identical orrelated parts of mRNA or DNA molecules.

The terms “oligonucleotides” or “nucleic acid probes” are prepared basedon the polynucleotide sequences provided in the present invention.Oligonucleotides comprise portions of such a polynucleotide sequencehaving at least about 15 nucleotides and usually at least about 20nucleotides. Nucleic acid probes comprise portions of such apolynucleotide sequence having fewer nucleotides than about 6 kb,usually fewer than about 1 kb. After appropriate testing to eliminatefalse positives, these probes may, for example, be used to determinewhether specific mRNA molecules are present in a cell or tissue or toisolate similar nucleic acid sequences from chromosomal DNA as describedby Walsh et al. (Walsh, P. S. et al., 1992, PCR Methods Appl 1:241-250).

The term “probes” includes naturally occurring or recombinant orchemically synthesized single- or double-stranded nucleic acids. Theymay be labeled by nick translation, Klenow fill-in reaction, PCR orother methods well known in the art. Probes of the present invention,their preparation and/or labeling are elaborated in Sambrook, J. et al.,1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols inMolecular Biology, John Wiley & Sons, New York N.Y., both of which areincorporated herein by reference in their entirety.

The term “stringent” is used to refer to conditions that are commonlyunderstood in the art as stringent. Stringent conditions can includehighly stringent conditions (i.e., hybridization to filter-bound DNAunder in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C., and washing in 0.1×SSC/0.1% SDS at 68° C.), and moderately stringentconditions (i.e., washing in 0.2×SSC/0.1% SDS at 42° C.).

In instances wherein hybridization of deoxyoligonucleotides isconcerned, additional exemplary stringent hybridization conditionsinclude washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-baseoligos), and 60° C. (for 23-base oligos).

The term “recombinant,” when used herein to refer to a polypeptide orprotein, means that a polypeptide or protein is derived from recombinant(e.g., microbial or mammalian) expression systems. “Microbial” refers torecombinant polypeptides or proteins made in bacterial or fungal (e.g.,yeast) expression systems. As a product, “recombinant microbial” definesa polypeptide or protein essentially free of native endogenoussubstances and unaccompanied by associated native glycosylation.Polypeptides or proteins expressed in most bacterial cultures, e.g., E.coli, will be free of glycosylation modifications; polypeptides orproteins expressed in yeast will have a glycosylation pattern in generaldifferent from those expressed in mammalian cells.

The term “recombinant expression vehicle or vector” refers to a plasmidor phage or virus or vector, for expressing a polypeptide from a DNA(RNA) sequence. An expression vehicle can comprise a transcriptionalunit comprising an assembly of (1) a genetic element or elements havinga regulatory role in gene expression, for example, promoters orenhancers, (2) a structural or coding sequence which is transcribed intomRNA and translated into protein, and (3) appropriate transcriptioninitiation and termination sequences. Structural units intended for usein yeast or eukaryotic expression systems preferably include a leadersequence enabling extracellular secretion of translated protein by ahost cell. Alternatively, where recombinant protein is expressed withouta leader or transport sequence, it may include an N-terminal methionineresidue. This residue may or may not be subsequently cleaved from theexpressed recombinant protein to provide a final product.

The term “recombinant expression system” means host cells which havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit extrachromosomally.Recombinant expression systems as defined herein will expressheterologous polypeptides or proteins upon induction of the regulatoryelements linked to the DNA segment or synthetic gene to be expressed.This term also means host cells which have stably integrated arecombinant genetic element or elements having a regulatory role in geneexpression, for example, promoters or enhancers. Recombinant expressionsystems as defined herein will express polypeptides or proteinsendogenous to the cell upon induction of the regulatory elements linkedto the endogenous DNA segment or gene to be expressed. The cells can beprokaryotic or eukaryotic.

The term “open reading frame,” ORF, means a series of nucleotidetriplets coding for amino acids without any termination codons and is asequence translatable into protein.

The term “expression modulating fragment,” EMF, means a series ofnucleotides which modulates the expression of an operably linked ORF oranother EMF.

As used herein, a sequence is said to “modulate the expression of anoperably linked sequence” when the expression of the sequence is alteredby the presence of the EMF. EMFs include, but are not limited to,promoters, and promoter modulating sequences (inducible elements). Oneclass of EMFs are fragments which induce the expression or an operablylinked ORF in response to a specific regulatory factor or physiologicalevent.

As used herein, an “uptake modulating fragment,” UMF, means a series ofnucleotides which mediate the uptake of a linked DNA fragment into acell. UMFs can be readily identified using known UMFs as a targetsequence or target motif with the computer-based systems describedbelow.

The presence and activity of a UMF can be confirmed by attaching thesuspected UMF to a marker sequence. The resulting nucleic acid moleculeis then incubated with an appropriate host under appropriate conditionsand the uptake of the marker sequence is determined. As described above,a UMF will increase the frequency of uptake of a linked marker sequence.

The term “active” refers to those forms of the polypeptide which retainthe biologic and/or immunologic activities of any naturally occurringpolypeptide.

The term “naturally occurring polypeptide” refers to polypeptidesproduced by cells that have not been genetically engineered andspecifically contemplates various polypeptides. arising frompost-translational modifications of the polypeptide including, but notlimited to, acetylation, carboxylation, glycosylation, phosphorylation,lipidation and acylation.

The term “derivative” refers to polypeptides chemically modified by suchtechniques as ubiquitination, labeling (e.g., with radionuclides orvarious enzymes), pegylation (derivatization with polyethylene glycol)and insertion or substitution by chemical synthesis of amino acids suchas ornithine, which do not normally occur in human proteins.

The term “recombinant variant” refers to any polypeptide differing fromnaturally occurring polypeptides by amino acid insertions, deletions,and substitutions, created using recombinant DNA techniques. Guidance indetermining which amino acid residues may be replaced, added or deletedwithout abolishing activities of interest, such as cellular trafficking,may be found by comparing the sequence of the particular polypeptidewith that of homologous peptides and minimizing the number of amino acidsequence changes made in regions of high homology.

Preferably, amino acid “substitutions” are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, i.e., conservative amino acid replacements. Aminoacid substitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid. “Insertions” or“deletions” are typically in the range of about 1 to 5 amino acids. Thevariation allowed may be experimentally determined by systematicallymaking insertions, deletions, or substitutions of amino acids in apolypeptide molecule using recombinant DNA techniques and assaying theresulting recombinant variants for activity.

Alternatively, where alteration of function is desired, insertions,deletions or non-conservative alterations can be engineered to producealtered polypeptides. Such alterations can, for example, alter one ormore of the biological functions or biochemical characteristics of thepolypeptides of the invention. For example, such alterations may changepolypeptide characteristics such as ligand-binding affinities,interchain affinities, or degradation/turnover rate. Further, suchalterations can be selected so as to generate polypeptides that arebetter suited for expression, scale up and the like in the host cellschosen for expression. For example, cysteine residues can be deleted orsubstituted with another amino acid residue in order to eliminatedisulfide bridges.

As used herein, “substantially equivalent” can refer both to nucleotideand amino acid sequences, for example a mutant sequence, that variesfrom a reference sequence by one or more substitutions, deletions, oradditions, the net effect of which does not result in an adversefunctional dissimilarity between the reference and subject sequences.Typically, such a substantially equivalent sequence varies from one ofthose listed herein by no more than about 20% (i.e., the number ofindividual residue substitutions, additions, and/or deletions in asubstantially equivalent sequence, as compared to the correspondingreference sequence, divided by the total number of residues in thesubstantially equivalent sequence is about 0.2 or less). Such a sequenceis said to have 80% sequence identity to the listed sequence. In oneembodiment, a substantially equivalent, e.g., mutant, sequence of theinvention varies from a listed sequence by no more than 10% (90%sequence identity); in a variation of this embodiment, by no more than5% (95% sequence identity); and in a further variation of thisembodiment, by no more than 2% (98% sequence identity). Substantiallyequivalent, e.g., mutant, amino acid sequences according to theinvention generally have at least 95% sequence identity with a listedamino acid sequence, whereas substantially equivalent nucleotidesequence of the invention can have lower percent sequence identities,taking into account, for example, the redundancy or degeneracy of thegenetic code. For the purposes of the present invention, sequenceshaving substantially equivalent biological activity and substantiallyequivalent expression characteristics are considered substantiallyequivalent. For the purposes of determining equivalence, truncation ofthe mature sequence (e.g., via a mutation which creates a spurious stopcodon) should be disregarded.

Nucleic acid sequences encoding such substantially equivalent sequences,e.g., sequences of the recited percent identities, can routinely beisolated and identified via standard hybridization procedures well knownto those of skill in the art.

Where desired, an expression vector may be designed. to contain a“signal or leader sequence” which will direct the polypeptide throughthe membrane of a cell. Such a sequence may be naturally present on thepolypeptides of the present invention or provided from heterologousprotein sources by recombinant DNA techniques.

A polypeptide “fragment,” “portion,” or “segment” is a stretch of aminoacid residues of at least about 5 amino acids, often at least about 7amino acids, typically at least about 9 to 13 amino acids, and, invarious embodiments, at least about 17 or more amino acids. To beactive, any polypeptide must have sufficient length to display biologicand/or immunologic activity. In a preferred embodiment, the IL-1Hy1fragment has a molecular weight as determined by SDS-PAGE of about 16kDa or less. More preferably, the IL-1Hy1 fragment has a molecular massof about 15.5 kDa.

Alternatively, recombinant variants encoding these same or similarpolypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsin the polynucleotide sequence may be reflected in the polypeptide ordomains of other peptides added to the polypeptide to modify theproperties of any part of the polypeptide, to change characteristicssuch as ligand-binding affinities, interchain affinities, ordegradation/turnover rate.

The term “activated” cells as used in this application are those whichare engaged in extracellular or intracellular membrane trafficking,including the export of neurosecretory or enzymatic molecules as part ofa normal or disease process.

The term “purified” as used herein denotes that the indicated nucleicacid or polypeptide is present in the substantial absence of otherbiological macromolecules, e.g., polynucleotides, proteins, and thelike. In one embodiment, the polynucleotide or polypeptide is purifiedsuch that it constitutes at least 95% by weight, more preferably atleast 99.8% by weight, of the indicated biological macromoleculespresent (but water, buffers, and other small molecules, especiallymolecules having a molecular weight of less than 1000 daltons, can bepresent).

The term “isolated” as used herein refers to a nucleic acid orpolypeptide separated from at least one other component (e.g., nucleicacid or polypeptide) present with the nucleic acid or polypeptide in itsnatural source. In one embodiment, the nucleic acid or polypeptide isfound in the presence of (if anything) only a solvent, buffer, ion, orother component normally present in a solution of the same. The terms“isolated” and “purified” do not encompass nucleic acids or polypeptidespresent in their natural source.

The term “infection” refers to the introduction of nucleic acids into asuitable host cell by use of a virus or viral vector.

The term “transformation” means introducing DNA into a suitable hostcell so that the DNA is replicable, either as an extrachromosomalelement, or by chromosomal integration.

The term “transfection” refers to the taking up of an expression vectorby a suitable host cell, whether or not any coding sequences are in factexpressed.

The term “intermediate fragment” means a nucleic acid between 5 and 1000bases in length, and preferably between 10 and 40 bp in length.

The term “secreted” includes a protein that is transported across orthrough a membrane, including transport as a result of signal sequencesin its amino acid sequence when it is expressed in a suitable host cell.“Secreted” proteins include without limitation proteins secreted wholly(e.g., soluble proteins) or partially (e.g., receptors) from the cell inwhich they are expressed. “Secreted” proteins also include withoutlimitation proteins which are transported across the membrane of theendoplasmic reticulum. “Secreted” proteins are also intended to includeproteins containing non-typical signal sequences (e.g. lnterleukin-1Beta, see Krasney, P. A. and Young, P. R. (1992) Cytokine 4(2): 134-143) and factors released from damaged cells (e.g. lnterleukin-1Receptor Antagonist, see Arend, W. P. et. al. (1998) Annu. Rev. Immunol.16:27-55)

Each of the above terms is meant to encompasses all that is describedfor each, unless the context dictates otherwise.

Nucleic Acids and Polypeptides of the Invention

Nucleotide and amino acid sequences of the invention are reported below.Fragments of the proteins of the present invention which are capable ofexhibiting biological activity are also encompassed by the presentinvention. Fragments of the protein may be in linear form or they may becyclized using known methods, for example, as described in H. U.Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S.McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both ofwhich are incorporated herein by reference. Such fragments may be fusedto carrier molecules such as immunoglobulins for many purposes,including increasing the valency of protein binding sites. For example,fragments of the protein may be fused through “linker” sequences to theFc portion of an immunoglobulin. For a bivalent form of the protein,such a fusion could be to the Fc portion of an IgG molecule. Otherimmunoglobulin isotypes may also be used to generate such fusions. Forexample, a protein-IgM fusion would generate a decavalent form of theprotein of the invention.

The present invention also provides both full-length and mature forms(for example, without a signal sequence or precursor sequence) of thedisclosed proteins. The full-length form of the such proteins isidentified in the sequence listing by translation of the nucleotidesequence of each disclosed clone. The mature form of such protein may beobtained by expression of the disclosed full-length polynucleotide in asuitable mammalian cell or other host cell. The sequence of the matureform of the protein is also determinable from the amino acid sequence ofthe full-length form. Where protein of the present invention is membranebound, soluble forms of the protein are also provided. In such formspart or all of the regions causing the protein to be membrane bound aredeleted so that the protein is fully secreted from the cell in which itis expressed.

The present invention also provides genes corresponding to the cDNAsequences disclosed herein. The corresponding genes can be isolated inaccordance with known methods using the sequence information disclosedherein. Such methods include the preparation of probes or primers fromthe disclosed sequence information for identification and/oramplification of genes in appropriate genomic libraries or other sourcesof genomic materials. Species homologs of the disclosed polynucleotidesand proteins are also provided by the present invention. Specieshomologs may be isolated and identified by making suitable probes orprimers from the sequences provided herein and screening a suitablenucleic acid source from the desired species. The invention alsoencompasses allelic variants of the disclosed polynucleotides orproteins; that is, naturally-occurring alternative forms of the isolatedpolynucleotide which also encode proteins which are identical,homologous or related to that encoded by the polynucleotides. Thecompositions of the present invention include isolated polynucleotides,including recombinant DNA molecules, cloned genes or degenerate variantsthereof, especially naturally occurring variants such as allelicvariants, novel isolated polypeptides, and antibodies that specificallyrecognize one or more epitopes present on such polypeptides. Specieshomologs of the disclosed polynucleotides and proteins are also providedby the present invention. Species homologs may be isolated andidentified by making suitable probes or primers from the sequencesprovided herein and screening a suitable nucleic acid source from thedesired species. The invention also encompasses allelic variants of thedisclosed polynucleotides or proteins; that is, naturally-occurringalternative forms of the isolated polynucleotide which also encodeproteins which are identical, homologous or related to that encoded bythe polynucleotides.

6.2 Nucleic Acids of the Invention

The isolated polynucleotides of the invention include, but are notlimited to, a polynucleotide encoding a polypeptide comprising the aminoacid sequence of SEQ ID NOS: 3 or 5.

The isolated polynucleotides of the invention further include, but arenot limited to a polynucleotide comprising the nucleotide sequence ofSEQ ID NOS: 1, 2, 4, or 6; a polynucleotide comprising the full lengthprotein coding sequence of SEQ ID NOS: 1, 2, 4, or 6, and; apolynucleotide comprising the nucleotide sequence of the mature proteincoding sequence of SEQ ID NOS: 1, 2, 4, or 6. The polynucleotides of thepresent invention also include, but are not limited to, a polynucleotidethat hybridizes to the complement of the nucleotide sequence of SEQ IDNOS: 1, 2, 4, or 6 under stringent hybridization conditions; apolynucleotide which is an allelic variant of any polynucleotide recitedabove; a polynucleotide which encodes a species homolog of any of theproteins recited above; or a polynucleotide that encodes a polypeptidecomprising a specific domain or truncation of the polypeptide of SEQ IDNOS: 1, 2, 4, or 6.

The isolated polynucleotides of the invention further include, but arenot limited to a polynucleotide comprising the nucleotide sequence ofthe genomic clone SEQ ID NOS: 7 or 8; a polynucleotide assembled fromone or more of the exons of SEQ ID NOS: 7 or 8 (e.g., alternativesplicing); a polynucleotide assembled from one or more of the introns ofSEQ ID NOS: 7 or 8; a polynucleotide assembled from one or more of theexons of SEQ ID NOS: 7 or 8 and one or more of the introns of SEQ IDNOS: 7 or 8; a polynucleotide comprising the full length protein codingsequence of SEQ ID NOS: 7 or 8; a polynucleotide comprising thenucleotide sequence of the mature protein coding sequence of SEQ ID NOS:7 or 8.

The polynucleotides of the present invention also include, but are notlimited to, a polynucleotide that hybridizes to the complement of thenucleotide sequence of SEQ ID NOS: 7 or 8 under stringent hybridizationconditions; a polynucleotide that hybridizes to the complement of anyone of the introns or exons of SEQ ID NOS: 7 or 8 under stringenthybridization conditions; a polynucleotide which is an allelic variantof any polynucleotide recited above; a polynucleotide which encodes aspecies homolog of any of the proteins recited above; or apolynucleotide that encodes a polypeptide comprising a specific domainor truncation of the polypeptide of SEQ ID NOS: 7 or 8.

The polynucleotides of the present invention still further include, butare not limited to, a polynucleotide comprising the nucleotide sequenceof the cDNA insert of clone pIL-1Hy273 deposited with the American TypeCulture Collection (ATCC; 10801 University Blvd., Manassas, Va.,20110-2209, U.S.A.); a polynucleotide comprising a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 3 or 5 whichpolynucleotide is assembled from the cDNA insert of clone pIL-1Hy273; apolynucleotide comprising the full length protein coding sequence of SEQID NO: 3 or 5 which polynucleotide is assembled from the cDNA insert ofclone pIL-1Hy273;or, a polynucleotide comprising the nucleotide sequenceof the mature protein coding sequence of SEQ ID NO: 3 or 5.

The polynucleotides of the invention additionally include the complementof any of the polynucleotides recited above.

The polynucleotides of the invention also provide polynucleotidesincluding nucleotide sequences that are substantially equivalent to thepolynucleotides recited above. Polynucleotides according to theinvention can have at least about 80%, more typically at least about90%, and even more typically at least about 95%, sequence identity to apolynucleotide recited above. The invention also provides the complementof the polynucleotides including a nucleotide sequence that has at leastabout 80%, more typically at least about 90%, and even more typically atleast about 95%, sequence identity to a polynucleotide encoding apolypeptide recited above. The polynucleotide can be DNA (genomic, cDNA,amplified, or synthetic) or RNA. Methods and algorithms for obtainingsuch polynucleotides are well known to those of skill in the art and caninclude, for example, methods for determining hybridization conditionswhich can routinely isolate polynucleotides of the desired sequenceidentities.

A polynucleotide according to the invention can be joined to any of avariety of other nucleotide sequences by well-established recombinantDNA techniques (see Sambrook J et al. (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, NY). Useful nucleotidesequences for joining to polypeptides include an assortment of vectors,e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and thelike, that are well known in the art. Accordingly, the invention alsoprovides a vector including a polynucleotide of the invention and a hostcell containing the polynucleotide. In general, the vector contains anorigin of replication functional in at least one organism, convenientrestriction endonuclease sites, and a selectable marker for the hostcell. Vectors according to the invention include expression vectors,replication vectors, probe generation vectors, and sequencing vectors. Ahost cell according to the invention can be a prokaryotic or eukaryoticcell and can be a unicellular organism or part of a multicellularorganism.

The polynucleotides of the present invention also make possible thedevelopment, through, e.g., homologous recombination or knock outstrategies, of animals that fail to express functional IL-1Hy1 or thatexpress a variant of IL-1Hy1. Such animals are useful as models forstudying the in vivo activities of IL-1Hy1 as well as for studyingmodulators of IL-1Hy1.

In preferred methods to determine biological functions of thepolypeptides of the invention in vivo, one or more genes provided by theinvention are either over expressed or inactivated in the germ line ofanimals using homologous recombination [Capecchi, Science 244:1288-1292(1989)]. Animals in which the gene is over expressed, under theregulatory control of exogenous or endogenous promoter elements, areknown as transgenic animals. Animals in which an endogenous gene hasbeen inactivated by homologous recombination are referred to as“knockout” animals. Knockout animals, preferably non-human mammals, canbe prepared as described in U.S. Pat. No. 5,557,032, incorporated hereinby reference. Transgenic animals are useful to determine the rolespolypeptides of the invention play in biological processes, andpreferably in disease states. Transgenic animals are useful as modelsystems to identify compounds that modulate lipid metabolism. Transgenicanimals, preferably non-human mammals, are produced using methods asdescribed in U.S. Pat. No. 5,489,743 and PCT Publication No. WO94/28122,incorporated herein by reference.

Transgenic animals can be prepared wherein all or part of apolynucleotides of the invention promoter is either activated orinactivated to alter the level of expression of the polypeptides of theinvention. Inactivation can be carried out using homologousrecombination methods described above. Activation can be achieved bysupplementing or even replacing the homologous promoter to provide forincreased protein expression. The homologous promoter can besupplemented by insertion of one or more heterologous enhancer elementsknown to confer promoter activation in a particular tissue.

Knowledge of IL-1Hy1 DNA sequences allows for modification of cells topermit, or increase, expression of endogenous IL-1Hy1. Cells can bemodified (e.g., by homologous recombination) to provide increasedIL-1Hy1 expression by replacing, in whole or in part, the naturallyoccurring IL-1Hy1 promoter with all or part of a heterologous promoterso that the cells express IL-1Hy1 at higher levels. The heterologouspromoter is inserted in such a manner that it is operatively linked toIL-1Hy1 encoding sequences. See, for example, PCT InternationalPublication No. WO94/12650, PCT International Publication No.WO92/20808, and PCT International Publication No. WO91/09955. It is alsocontemplated that, in addition to heterologous promoter DNA, amplifiablemarker DNA (e.g., ada, dhfr, and the multifunctional CAD gene whichencodes carbamyl phosphate synthase, aspartate transcarbamylase, anddihydroorotase) and/or intron DNA may be inserted along with theheterologous promoter DNA. If linked to the IL-1Hy1 coding sequence,amplification of the marker DNA by standard selection methods results inco-amplification of the IL-1Hy1 coding sequences in the cells.

The sequences falling within the scope of the present invention are notlimited to the specific sequences herein described, but also includeallelic variations thereof. Allelic variations can be routinelydetermined by comparing the sequence provided in SEQ ID NOS: 1, 2, 4, 6,7 or 8, a representative fragment thereof, or a nucleotide sequence atleast 99.9% identical to SEQ ID NOS: 1, 2, 4, 6, 7, or 8, with asequence from another isolate of the same species. Furthermore, toaccommodate codon variability, the invention includes nucleic acidmolecules coding for the same amino acid sequences as do the specificORFs disclosed herein. In other words, in the coding region of an ORF,substitution of one codon for another which encodes the same amino acidis expressly contemplated. Any specific sequence disclosed herein can bereadily screened for errors by resequencing a particular fragment, suchas an ORF, in both directions (i.e., sequence both strands).

The present invention further provides recombinant constructs comprisinga nucleic acid having the sequence of SEQ ID NOS: 1, 2, 4, 6, 7 or 8, ora fragment thereof. The recombinant constructs of the present inventioncomprise a vector, such as a plasmid or viral vector, into which anucleic acid having the sequence of SEQ ID NOS: 1, 2, 4, 6, 7 or 8 or afragment thereof is inserted, in a forward or reverse orientation. Inthe case of a vector comprising one of the ORFs of the presentinvention, the vector may further comprise regulatory sequences,including for example, a promoter, operably linked to the ORF. Forvectors comprising the EMFs and UMFs of the present invention, thevector may further comprise a marker sequence or heterologous ORFoperably linked to the EMF or UMF. Large numbers of suitable vectors andpromoters are known to those of skill in the art and are commerciallyavailable for generating the recombinant constructs of the presentinvention. The following vectors are provided by way of example.Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a,pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).

The isolated polynucleotide of the invention may be operably linked toan expression control sequence such as the pMT2 or pED expressionvectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490(1991), in order to produce the protein recombinantly. Many suitableexpression control sequences are known in the art. General methods ofexpressing recombinant proteins are also known and are exemplified in R.Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein“operably linked” means that the isolated polynucleotide of theinvention and an expression control sequence are situated within avector or cell in such a way that the protein is expressed by a hostcell which has been transformed (transfected) with the ligatedpolynucleotide/expression control sequence.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacZ, lacZ, T3, T7, gpt, lambda PR, and trc.Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-1.Selection of the appropriate vector and promoter is well within thelevel of ordinary skill in the art. Generally, recombinant expressionvectors will include origins of replication and selectable markerspermitting transformation of the host cell, e.g., the ampicillinresistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoterderived from a highly-expressed gene to direct transcription of adownstream structural sequence. Such promoters can be derived fromoperons encoding glycolytic enzymes such as 3-phosphoglycerate kinase(PGK), a-factor, acid phosphatase, or heat shock proteins, among others.The heterologous structural sequence is assembled in appropriate phasewith translation initiation and termination sequences, and preferably, aleader sequence capable of directing secretion of translated proteininto the periplasmic space or extracellular medium. Optionally, theheterologous sequence can encode a fusion protein including anN-terminal identification peptide imparting desired characteristics,e.g., stabilization or simplified purification of expressed recombinantproduct. Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed. Followingtransformation of a suitable host strain and growth of the host strainto an appropriate cell density, the selected promoter is induced orderepressed by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. Cells aretypically harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification.

Included within the scope of the nucleic acid sequences of the inventionare nucleic acid sequences that hybridize under stringent conditions toa fragment of the DNA sequence in FIG. 2, 5, 8, or 9 or its complement,which fragment is greater than about 10 bp, preferably 20-50 bp, andeven greater than 100 bp. In accordance with the invention,polynucleotide sequences which encode the novel nucleic acids, orfunctional equivalents thereof, may be used to generate recombinant DNAmolecules that direct the expression of that nucleic acid, or afunctional equivalent thereof, in appropriate host cells.

The nucleic acid sequences of the invention are further directed tosequences which encode variants of the described nucleic acids. Theseamino acid sequence variants may be prepared by methods known in the artby introducing appropriate nucleotide changes into a native or variantpolynucleotide. There are two variables in the construction of aminoacid sequence variants: the location of the mutation and the nature ofthe mutation. The amino acid sequence variants of the nucleic acids arepreferably constructed by mutating the polynucleotide to give an aminoacid sequence that does not occur in nature. These amino acidalterations can be made at sites that differ in the nucleic acids fromdifferent species (variable positions) or in highly conserved regions(constant regions). Sites at such locations will typically be modifiedin series, e.g., by substituting first with conservative choices (e.g.,hydrophobic amino acid to a different hydrophobic amino acid) and thenwith more distant choices (e.g., hydrophobic amino acid to a chargedamino acid), and then deletions or insertions may be made at the targetsite. Amino acid sequence deletions generally range from about 1 to 30residues, preferably about 1 to 10 residues, and are typicallycontiguous. Amino acid insertions include amino- and/orcarboxyl-terminal fusions ranging in length from one to one hundred ormore residues, as well as intrasequence insertions of single or multipleamino acid residues. Intrasequence insertions may range generally fromabout 1 to 10 amino residues, preferably from 1 to 5 residues. Examplesof terminal insertions include the heterologous signal sequencesnecessary for secretion or for intracellular targeting in different hostcells.

In a preferred method, polynucleotides encoding the novel nucleic acidsare changed via site-directed mutagenesis. This method usesoligonucleotide sequences that encode the polynucleotide sequence of thedesired amino acid variant, as well as a sufficient adjacent nucleotideon both sides of the changed amino acid to form a stable duplex oneither side of the site of being changed. In general, the techniques ofsite-directed mutagenesis are well known to those of skill in the artand this technique is exemplified by publications such as, Edelman etal., DNA 2:183 (1983). A versatile and efficient method for producingsite-specific changes in a polynucleotide sequence was published byZoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982). PCR may alsobe used to create amino acid sequence variants of the novel nucleicacids. When small amounts of template DNA are used as starting material,primer(s) that differs slightly in sequence from the correspondingregion in the template DNA can generate the desired amino acid variant.PCR amplification results in a population of product DNA fragments thatdiffer from the polynucleotide template encoding the polypeptide at theposition specified by the primer. The product DNA fragments replace thecorresponding region in the plasmid and this gives the desired aminoacid variant.

A further technique for generating amino acid variants is the cassettemutagenesis technique described in Wells et al., Gene 34:315 (1985); andother mutagenesis techniques well known in the art, such as, forexample, the techniques in Sambrook et al., supra, and Current Protocolsin Molecular Biology, Ausubel et al. Due to the inherent degeneracy ofthe genetic code, other DNA sequences which encode substantially thesame or a functionally equivalent amino acid sequence may be used in thepractice of the invention for the cloning and expression of these novelnucleic acids. Such DNA sequences include those which are capable ofhybridizing to the appropriate novel nucleic acid sequence understringent conditions.

Polynucleotides of the invention can also be used to induce immuneresponses. For example, as described in Fan et al., Nat. Biotech.17:870-872 (1999), incorporated herein by reference, nucleic acidsequences encoding a polypeptide may be used to generate antibodiesagainst the encoded polypeptide following topical administration ofnaked plasmid DNA or following injection, and preferably intramuscularinjection of the DNA. The nucleic acid sequences are preferably insertedin a recombinant expression vector and may be in the form of naked DNA.

6.3. Hosts

The present invention further provides host cells genetically engineeredto contain the polynucleotides of the invention. For example, such hostcells may contain nucleic acids of the invention introduced into thehost cell using known transformation, transfection or infection methods.The present invention still further provides host cells geneticallyengineered to express the polynucleotides of the invention, wherein suchpolynucleotides are in operative association with a regulatory sequenceheterologous to the host cell which drives expression of thepolynucleotides in the cell.

The host cell can be a higher eukaryotic host cell, such as a mammaliancell, a lower eukaryotic host cell, such as a yeast cell, or the hostcell can be a prokaryotic cell, such as a bacterial cell. Introductionof the recombinant construct into the host cell can be effected bycalcium phosphate transfection, DEAE, dextran mediated transfection, orelectroporation (Davis, L. et al., Basic Methods in Molecular Biology(1986)). The host cells containing one of polynucleotides of theinvention, can be used in conventional manners to produce the geneproduct encoded by the isolated fragment (in the case of an ORF) or canbe used to produce a heterologous protein under the control of the EMF.

Any host/vector system can be used to express one or more of the ORFs ofthe present invention. These include, but are not limited to, eukaryotichosts such as HeLa cells, Cv-1 cell, COS cells, and Sf9 cells, as wellas prokaryotic host such as E. coli and B. subtilis. The most preferredcells are those which do not normally express the particular polypeptideor protein or which expresses the polypeptide or protein at low naturallevel. Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al., inMolecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989), the disclosure of which is hereby incorporated byreference.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, for example, the C127, 3T3, CHO, HeLa and BHK cell tines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter and also any necessary ribosome binding sites,polyadenylation site, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking nontranscribed sequences. DNAsequences derived from the SV40 viral genome, for example, SV40 origin,early promoter, enhancer, splice, and polyadenylation sites may be usedto provide the required nontranscribed genetic elements. Recombinantpolypeptides and proteins produced in bacterial culture are usuallyisolated by initial extraction from cell pellets, followed by one ormore salting-out, aqueous ion exchange or size exclusion chromatographysteps. Protein refolding steps can be used, as necessary, in completingconfiguration of the mature protein. Finally, high performance liquidchromatography (HPLC) can be employed for final purification steps.Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents.

A number of types of cells may act as suitable host cells for expressionof the protein. Mammalian host cells include, for example, monkey COScells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, humanepidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, othertransformed primate cell lines, normal diploid cells, cell strainsderived from in vitro culture of primary tissue, primary explants, HeLacells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.

Alternatively, it may be possible to produce the protein in lowereukaryotes such as yeast or in prokaryotes such as bacteria. Potentiallysuitable yeast strains include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing heterologous proteins. Potentially suitablebacterial strains include Escherichia coli, Bacillus subtilis,Salmonella typhimurium, or any bacterial strain capable of expressingheterologous proteins. If the protein is made in yeast or bacteria, itmay be necessary to modify the protein produced therein, for example byphosphorylation or glycosylation of the appropriate sites, in order toobtain the functional protein. Such covalent attachments may beaccomplished using known chemical or enzymatic methods.

In another embodiment of the present invention, cells and tissues may beengineered to express an endogenous gene comprising the polynucleotidesof the invention under the control of inducible regulatory elements, inwhich case the regulatory sequences of the endogenous gene may bereplaced by homologous recombination. As described herein, genetargeting can be used to replace a gene's existing regulatory regionwith a regulatory sequence isolated from a different gene or a novelregulatory sequence synthesized by genetic engineering methods. Suchregulatory sequences may be comprised of promoters, enhancers,scaffold-attachment regions, negative regulatory elements,transcriptional initiation sites, regulatory protein binding sites orcombinations of said sequences. Alternatively, sequences which affectthe structure or stability of the RNA or protein produced may bereplaced, removed, added, or otherwise modified by targeting, includingpolyadenylation signals. mRNA stability elements, splice sites, leadersequences for enhancing or modifying transport or secretion propertiesof the protein, or other sequences which alter or improve the functionor stability of protein or RNA molecules.

The targeting event may be a simple insertion of the regulatorysequence, placing the gene under the control of the new regulatorysequence, e.g., inserting a new promoter or enhancer or both upstream ofa gene. Alternatively, the targeting event may be a simple deletion of aregulatory element, such as the deletion of a tissue-specific negativeregulatory element. Alternatively, the targeting event may replace anexisting element; for example, a tissue-specific enhancer can bereplaced by an enhancer that has broader or different cell-typespecificity than the naturally occurring elements. Here, the naturallyoccurring sequences are deleted and new sequences are added. In allcases, the identification of the targeting event may be facilitated bythe use of one or more selectable marker genes that are contiguous withthe targeting DNA, allowing for the selection of cells in which theexogenous DNA has integrated into the host cell genome. Theidentification of the targeting event may also be facilitated by the useof one or more marker genes exhibiting the property of negativeselection, such that the negatively selectable marker is linked to theexogenous DNA, but configured such that the negatively selectable markerflanks the targeting sequence, and such that a correct homologousrecombination event with sequences in the host cell genome does notresult in the stable integration of the negatively selectable marker.Markers useful for this purpose include the Herpes Simplex Virusthymidine kinase (TK) gene or the bacterial xanthine-guaninephosphoribosyl-transferase (gpt) gene.

The gene targeting or gene activation techniques which can be used inaccordance with this aspect of the invention are more particularlydescribed in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461to Sherwin et al.; International Application No. PCT/US92/09627(WO93/09222) by Selden et al.; and International Application No.PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which isincorporated by reference herein in its entirety.

6.4. Polypeptides of the Invention

SEQ ID NO: 2 encodes the polypeptide sequence of SEQ ID NO: 3. An aminoacid alignment of SEQ ID NO: 3 and rat, mouse, rabbit and humanInterleukin-1 receptor antagonist polypeptides is shown FIG. 1. SEQ IDNO: 3 displays significant (71%) amino acid homology with humanInterleukin-1 receptor antagonist, and thus, represents a novel moleculethat is highly related to, yet distinct from Interleukin-1 receptorantagonist. This is the first cloned sequence of an Interleukin-1receptor antagonist related gene. This discovery of SEQ ID NO: 3indicates that a family of Interleukin-1 receptor antagonist relatedgenes exists. Additional family members can be identified using SEQ IDNO: 1 or 2 as a molecular probe. SEQ ID NO: 3 encodes a protein thatcontains a 4 amino acid insertion differing from all other previouslycharacterized Interleukin-1 receptor antagonists sequences (FIG. 1).This insertion may provide SEQ ID NO: 3 with a novel and previouslyuncharacterized activity. Likewise, FIG. 7 presents an amino acidalignment of SEQ ID NO: 5 with the cytoplasmic form of human IL-1 Ra(labeled “HUMIL1RASIC”). The alignment reveals a high degree of homologybetween the two; 48% of the amino acids were identical and 54% representconservative amino acid substitutions.

Interleukin-1 has pleiotropic biological activities many of whichadversely affect the organism, it would be expected that the moleculemust be tightly regulated if it is not to be injurious. Indeed, thereare several reports of Interleukin-1 inhibitors that regulate the actionof Interleukin-1. Interleukin-1 inhibitory activity has been reported inmonocyte conditioned medium, wherein the monocytes are grown on adherentimmune complexes. Arena, W. P., et al., 1985, Journal of Immun.,134:3868. Additionally, an inhibitor has been reported to be presenturine. Seckinger, P., et al., 1987, Journal of Immun., 139:1546. Lastly,a protein inhibitor, purified and cloned, that has interleukin-1receptor antagonist activity has been reported. Hannum, et at., 1990,Nature, 343:336, and Eisenberg, S., et al., 1990, Nature, 343:341.

It is thought that the Interleukin-1 inhibitor present in urine, andwhich has been partially purified and characterized by Seckinger, P. etal., and Seckinger, P., et al., 1987, Journal of Immun., 139:1541 issimilar, if not identical to the cloned Interleukin-1 receptorantagonist reported by Eisenberg, S., et al. (1990), Nature, 343:341;and Carter, D., et al (1990), Nature, 344:633.

Interleukin-1 receptor antagonist is a naturally occurring peptidesecreted by macrophages in response to many of the same stimuli whichcause the secretion of Interleukin-1 itself. Interleukin-1 receptorantagonist is a naturally occurring antagonist to the cytokines andrecognizes receptors on various cell types and blocks Interleukin-1mediated responses by occupying the receptor. (Wakabayashi et al., FASEBJ 1991;5:338; Okusawa et al. J Clin Invest 1988;81:1162; Ohlsson et al.,Nature 1990;348:550; Aiura, et al. Cytokine 1991;4:498; Fischer et al.Am J Physiol 1991;261:R442). In humans, Interleukin-1 receptorantagonist is a naturally occurring group of molecules; three forms havebeen characterized (two glycosylated and one non-glycosylated).

Fischer et al. (Am J Physiol 1991;261:R442) demonstrated that theadministration of a naturally occurring antagonist to Interleukin-1 willsignificantly blunt the cytokine cascade and improve survival in baboonsgiven a lethal dose of live bacteria. Interleukin-1 receptor antagonistsignificantly attenuates the decrease in mean arterial pressure andcardiac output and improves survival for severe acute pancreatitis.(U.S. Pat. No. 5,508,262) The systemic Interleukin-1 response observedas a result of bacterial sepsis was also diminished significantly,correlating with a decrease in the systemic response to bacterialsepsis.

Studies by Aiura et al.(Cytokine 1991 ;4:498) have shown thatInterleukin-1 receptor antagonist is protective in a rabbit model ofhypotensive gram-positive septic shock. The administration ofInterleukin-1 receptor antagonist in this animal model has been shown tomaintain mean arterial pressure compared to control, as well asdecreasing lung water and maintaining urine output. This workdemonstrated the role of Interleukin-1 and the protective role ofInterleukin-1 receptor antagonist in gram-positive septic shock.Interleukin-1 is the principal mediator in a patient's clinical responseto multiple different stresses regardless of the etiology (includingacute pancreatitis, sepsis, endotoxin shock, and cytokine inducedshock).

The isolated polypeptides of the invention include, but are not limitedto, a polypeptide comprising the amino acid sequence of SEQ ID NOS: 3 or5; a full length protein coding sequence of SEQ ID NOS: 3 or 5; a matureprotein coding sequence of SEQ ID NOS: 3 or 5, or a polypeptide encodedby one or more of the exons of SEQ ID NOS: 7 or 8.

The polypeptides of the present invention further include, but are notlimited to, a polypeptide comprising the amino acid sequence encoded bythe cDNA insert of clone pIL-1Hy273 deposited with the American TypeCulture Collection (ATCC; 10801 University Blvd., Manassas, Va.,20110-2209, U.S.A.); a full length protein of SEQ ID NO: 3 or 5assembled from the amino acid sequence encoded by the cDNA insert ofclone pIL-1Hy273; or, a mature protein coding sequence of SEQ ID NO: 3or 5 assembled from the amino acid sequence encoded by cDNA insert ofclone pIL-1Hy273.

Protein compositions of the present invention may further comprise anacceptable carrier, such as a hydrophilic, e.g., pharmaceuticallyacceptable, carrier.

The invention also relates to methods for producing a polypeptidecomprising growing a culture of the cells of the invention in a suitableculture medium, and purifying the protein from the culture. For example,the methods of the invention include a process for producing apolypeptide in which a host cell containing a suitable expression vectorthat includes a polynucleotide of the invention is cultured underconditions that allow expression of the encoded polypeptide. Thepolypeptide can be recovered from the culture, conveniently from theculture medium, and further purified. Preferred embodiments includethose in which the protein produced by such process is a full length ormature form of the protein.

The invention further provides a polypeptide including an amino acidsequence that is substantially equivalent to SEQ ID NOS: 3 or 5.Polypeptides according to the invention can have at least about 95%, andmore typically at least about 98%, sequence identity to SEQ ID NOS: 3 or5.

The present invention further provides isolated polypeptides encoded bythe nucleic acid fragments of the present invention or by degeneratevariants of the nucleic acid fragments of the present invention. By“degenerate variant” is intended nucleotide fragments which differ froma nucleic acid fragment of the present invention (e.g., an ORF) bynucleotide sequence but, due to the degeneracy of the genetic code,encode an identical polypeptide sequence. Preferred nucleic acidfragments of the present invention are the ORFs that encode proteins. Avariety of methodologies known in the art can be utilized to obtain anyone of the isolated polypeptides or proteins of the present invention.At the simplest level, the amino acid sequence can be synthesized usingcommercially available peptide synthesizers. This is particularly usefulin producing small peptides and fragments of larger polypeptides.Fragments are useful, for example, in generating antibodies against thenative polypeptide. In an alternative method, the polypeptide or proteinis purified from bacterial cells which naturally produce the polypeptideor protein. One skilled in the art can readily follow known methods forisolating polypeptides and proteins in order to obtain one of theisolated polypeptides or proteins of the present invention. Theseinclude, but are not limited to, immunochromatography, HPLC,size-exclusion chromatography, ion-exchange chromatography, andimmuno-affinity chromatography. See, e.g., Scopes, Protein Purification:Principles and Practice, Springer-Verlag (1994); Sambrook, et al., inMolecular Cloning: A Laboratory Manual; Ausubel et al., CurrentProtocols in Molecular Biology.

The polypeptides and proteins of the present invention can alternativelybe purified from cells which have been altered to express the desiredpolypeptide or protein. As used herein, a cell is said to be altered toexpress a desired polypeptide or protein when the cell, through geneticmanipulation, is made to produce a polypeptide or protein which itnormally does not produce or which the cell normally produces at a lowerlevel. One skilled in the art can readily adapt procedures forintroducing and expressing either recombinant or synthetic sequencesinto eukaryotic or prokaryotic cells in order to generate a cell whichproduces one of the polypeptides or proteins of the present invention.The purified polypeptides can be used in in vitro binding assays whichare well known in the art to identify molecules which bind to thepolypeptides. These molecules include but are not limited to, for e.g.,small molecules, molecules from combinatorial libraries, antibodies orother proteins. The molecules identified in the binding assay are thentested for antagonist or agonist activity in in vivo tissue culture oranimal models that are well known in the art. In brief, the moleculesare titrated into a plurality of cell cultures or animals and thentested for either cell/animal death or prolonged survival of theanimal/cells.

In addition, the binding molecules may be complexed with toxins, e.g.,ricin or cholera, or with other compounds that are toxic to cells. Thetoxin-binding molecule complex is then targeted to a tumor or other cellby the specificity of the binding molecule for SEQ ID NO: 3 or 5.

The protein of the invention may also be expressed as a product oftransgenic animals, e.g., as a component of the milk of transgenic cows,goats, pigs, or sheep which are characterized by somatic or germ cellscontaining a nucleotide sequence encoding the protein.

The protein may also be produced by known conventional chemicalsynthesis. Methods for constructing the proteins of the presentinvention by synthetic means are known to those skilled in the art. Thesynthetically constructed protein sequences, by virtue of sharingprimary, secondary or tertiary structural and/or conformationalcharacteristics with proteins may possess biological properties incommon therewith, including protein activity. Thus, they may be employedas biologically active or immunological substitutes for natural,purified proteins in screening of therapeutic compounds and inimmunological processes for the development of antibodies.

The proteins provided herein also include proteins characterized byamino acid sequences similar to those of purified proteins but intowhich modification are naturally provided or deliberately engineered.For example, modifications in the peptide or DNA sequences can be madeby those skilled in the art using known techniques. Modifications ofinterest in the protein sequences may include the alteration,substitution, replacement, insertion or deletion of a selected aminoacid residue in the coding sequence. For example, one or more of thecysteine residues may be deleted or replaced with another amino acid toalter the conformation of the molecule. Techniques for such alteration,substitution, replacement, insertion or deletion are well known to thoseskilled in the art (see, e.g., U.S. Pat. No. 4,518,584). Preferably,such alteration, substitution, replacement, insertion or deletionretains the desired activity of the protein.

Other fragments and derivatives of the sequences of proteins which wouldbe expected to retain protein activity in whole or in part and may thusbe useful for screening or other immunological methodologies may also beeasily made by those skilled in the art given the disclosures herein.Such modifications are believed to be encompassed by the presentinvention.

The protein may also be produced by operably linking the isolatedpolynucleotide of the invention to suitable control sequences in one ormore insect expression vectors, and employing an insect expressionsystem. Materials and methods for baculovirus/insect cell expressionsystems are commercially available in kit form from, e.g., Invitrogen,San Diego, Calif., U.S.A. (the MAXBAT™ kit), and such methods are wellknown in the art, as described in Summers and Smith, Texas AgriculturalExperiment Station Bulletin No. 1555 (1987), incorporated herein byreference. As used herein, an insect cell capable of expressing apolynucleotide of the present invention is “transformed.”

The protein of the invention may be prepared by culturing transformedhost cells under culture conditions suitable to express the recombinantprotein. The resulting expressed protein may then be purified from suchculture (i.e., from culture medium or cell extracts) using knownpurification processes, such as gel filtration and ion exchangechromatography. The purification of the protein may also include anaffinity column containing agents which will bind to the protein; one ormore column steps over such affinity resins as concanavalin A-agarose,HEPARIN-TOYOPEARL™. or CIBACROM BLUE 3GA SEPHAROSE™; one or more stepsinvolving hydrophobic interaction chromatography using such resins asphenyl ether, butyl ether, or propyl ether; or immunoaffinitychromatography.

Alternatively, the protein of the invention may also be expressed in aform which will facilitate purification. For example, it may beexpressed as a fusion protein, such as those of maltose binding protein(MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits forexpression and purification of such fusion proteins are commerciallyavailable from New England BioLab (Beverly, Mass.), Pharmacia(Piscataway, N.J.) and In Vitrogen, respectively. The protein can alsobe tagged with an epitope and subsequently purified by using a specificantibody directed to such epitope. One such epitope (“Flag”) iscommercially available from Kodak (New Haven, Conn.).

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the protein. Some or all of the foregoingpurification steps, in various combinations, can also be employed toprovide a substantially homogeneous isolated recombinant protein. Theprotein thus purified is substantially free of other mammalian proteinsand is defined in accordance with the present invention as an “isolatedprotein.”

The polypeptides of the invention include lnterleukin-1 ReceptorAntagonist (IL-1 RA) analogs. This embraces fragments of IL-1 RA of theinvention, as well as Interleukin-1 Receptor Antagonists which compriseone or more amino acids deleted, inserted, or substituted. Also, analogsof Interleukin-1 Receptor Antagonist of the invention embrace fusions ofInterleukin-1 Receptor Antagonist or modifications of Interleukin-1Receptor Antagonist, wherein the Interleukin-1 Receptor Antagonist oranalog is fused to another moiety or moieties, e.g., targeting moiety oranother therapeutic agent. Such analogs may exhibit improved propertiessuch as activity and/or stability. Examples of moieties which may befused to Interleukin-1 Receptor Antagonist or an analog include, forexample, targeting moieties which provide for the delivery ofpolypeptide to pancreatic cells, e.g., antibodies to pancreatic cells,antibodies to immune cells such as T-cells, monocytes, dendritic cells,granulocytes, etc., as well as receptor and ligands expressed onpancreatic or immune cells. Other moieties which may be fused toInterleukin-1 Receptor Antagonist include therapeutic agents which areused for treatment, for example, immunosuppressive drugs such ascyclosporin, SK506, azathioprine, CD3 antibodies and steroids. Also,Interleukin-1 Receptor Antagonist may be fused to immunostimulants,immune modulators, and other cytokines such as alpha or beta interferon.

6.5 Gene Therapy

Mutations in the IL-1Hy1 gene that result in loss of normal function ofthe IL-1Hy1 gene product underlie IL-1Hy1-related human disease states.The invention comprehends gene therapy to restore normal IL-1Hy1activity or to treating those disease states involving IL-1Hy1. Deliveryof a functional IL-1Hy1 gene to appropriate cells is effected ex vivo,in situ, or in vivo by use of vectors, and more particularly viralvectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), orex vivo by use of physical DNA transfer methods (e.g., liposomes orchemical treatments). See, for example, Anderson, Nature, supplement tovol. 392, no 6679, pp. 25-30 (1998). For additional reviews of genetherapy technology, see Friedmann, Science, 244: 1275-1281 (1989);Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357:455-460 (1992).

Introduction of any one of the nucleotides of the present invention or agene encoding the polypeptides of the present invention can also beaccomplished with extrachromosomal substrates (transient expression) orartificial chromosomes (stable expression). Cells may also be culturedex vivo in the presence of proteins of the present invention in order toproliferate or to produce a desired effect on or activity in such cells.Treated cells can then be introduced in vivo for therapeutic purposes.Alternatively, it is contemplated that in other human disease states,preventing the expression of or inhibiting the activity of IL-1Hy1 willbe useful in treating the disease states. It is contemplated thatantisense therapy or gene therapy could be applied to negativelyregulate the expression of polypeptides of the invention. Other methodsinhibiting expression of a protein include the introduction of antisensemolecules to the nucleic acids of the present invention, theircomplements, or their translated RNA sequences, by methods known in theart, the removal of the nucleic acids of the present invention such asusing targeted deletion methods, or the insertion of a negativeregulatory element such as a silencer, which is tissue specific.Further, the polypeptides of the present invention can be inhibited bythe introduction of antisense molecules that hybridize to nucleic acidsthat encode for the polypeptides of the present invention and by theremoval of a gene that encode for the polypeptides of the presentinvention.

The present invention still further provides cells geneticallyengineered in vivo to express the polynucleotides of the invention,wherein such polynucleotides are in operative association with aregulatory sequence heterologous to the host cell which drivesexpression of the polynucleotides in the cell. These methods can be usedto increase or decrease the expression of the polynucleotides of thepresent invention.

Knowledge of DNA sequences provided by the invention allows formodification of cells to permit, increase, or decrease, expression ofendogenous polypeptide. Cells can be modified (e.g., by homologousrecombination) to provide increased polypeptide expression by replacing,in whole or in part, the naturally occurring promoter with all or partof a heterologous promoter so that the cells express the protein athigher levels. The heterologous promoter is inserted in such a mannerthat it is operatively linked to the desired protein encoding sequences.See, for example, PCT International Publication No. WO 94/12650, PCTInternational Publication No. WO 92/20808, and PCT InternationalPublication No. WO 91/09955. It is also contemplated that, in additionto heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr,and the multifunctional CAD gene which encodes carbamyl phosphatesynthase, aspartate transcarbamylase, and dihydroorotase) and/or intronDNA may be inserted along with the heterologous promoter DNA. If linkedto the desired protein coding sequence, amplification of the marker DNAby standard selection methods results in co-amplification of the desiredprotein coding sequences in the cells.

In another embodiment of the present invention, cells and tissues may beengineered to express an endogenous gene comprising the polynucleotidesof the invention under the control of inducible regulatory elements, inwhich case the regulatory sequences of the endogenous gene may bereplaced by homologous recombination. As described herein, genetargeting can be used to replace a gene's existing regulatory regionwith a regulatory sequence isolated from a different gene or a novelregulatory sequence synthesized by genetic engineering methods. Suchregulatory sequences may be comprised of promoters, enhancers,scaffold-attachment regions, negative regulatory elements,transcriptional initiation sites, regulatory protein binding sites orcombinations of said sequences. Alternatively, sequences which affectthe structure or stability of the RNA or protein produced may bereplaced, removed, added, or otherwise modified by targeting. Thesesequence include polyadenylation signals, mRNA stability elements,splice sites, leader sequences for enhancing or modifying transport orsecretion properties of the protein, or other sequences which alter orimprove the function or stability of protein or RNA molecules.

The targeting event may be a simple insertion of the regulatorysequence, placing the gene under the control of the new regulatorysequence, e.g., inserting a new promoter or enhancer or both upstream ofa gene. Alternatively, the targeting event may be a simple deletion of aregulatory element, such as the deletion of a tissue-specific negativeregulatory element. Alternatively, the targeting event may replace anexisting element; for example, a tissue-specific enhancer can bereplaced by an enhancer that has broader or different cell-typespecificity than the naturally occurring elements. Here, the naturallyoccurring sequences are deleted and new sequences are added. In allcases, the identification of the targeting event may be facilitated bythe use of one or more selectable marker genes that are contiguous withthe targeting DNA, allowing for the selection of cells in which theexogenous DNA has integrated into the cell genome. The identification ofthe targeting event may also be facilitated by the use of one or moremarker genes exhibiting the property of negative selection, such thatthe negatively selectable marker is linked to the exogenous DNA, butconfigured such that the negatively selectable marker flanks thetargeting sequence, and such that a correct homologous recombinationevent with sequences in the host cell genome does not result in thestable integration of the negatively selectable marker. Markers usefulfor this purpose include the Herpes Simplex Virus thymidine kinase (TK)gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt)gene.

The gene targeting or gene activation techniques which can be used inaccordance with this aspect of the invention are more particularlydescribed in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461to Sherwin et al.; International Application No. PCT/US92/09627(WO93/09222) by Selden et al.; and International Application No.PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which isincorporated by reference herein in its entirety.

6.6. Deposit of Clone

The following clone, pIL-1Hy 273 was deposited with the American TypeCulture Collection (ATCC) 10801 University Avenue, Manassas, Va., onMar. 12, 1999 under the terms of the Budapest Treaty. The 2,648 basepair cDNA insert of clone pIL-1Hy 273 is contained in vector pSPORT1 andis flanked by Notl and Sall restriction sites. The clone represent aplasmid clone as described in the Examples set forth below.

Microorganism/Clone ATCC Accession No. pIL-1Hy273 203841

6.7. Uses and Biological Activity

The polynucleotides and proteins of the present invention are expectedto exhibit one or more of the uses or biological activities (includingthose associated with assays cited herein) identified below. Uses oractivities described for proteins of the present invention may beprovided by administration or use of such proteins or by administrationor use of polynucleotides encoding such proteins (such as, for example,in gene therapies or vectors suitable for introduction of DNA).

6.7.1. Research Uses and Utilities

The polynucleotides provided by the present invention can be used by theresearch community for various purposes. The polynucleotides can be usedto express recombinant protein for analysis, characterization ortherapeutic use; as markers for tissues in which the correspondingprotein is preferentially expressed (either constitutively or at aparticular stage of tissue differentiation or development or in diseasestates); as molecular weight markers on Southern gels; as chromosomemarkers or tags (when labeled) to identify chromosomes or to map relatedgene positions; to compare with endogenous DNA sequences in patients toidentify potential genetic disorders; as probes to hybridize and thusdiscover novel, related DNA sequences; as a source of information toderive PCR primers for genetic fingerprinting; as a probe to“subtract-out” known sequences in the process of discovering other novelpolynucleotides; for selecting and making oligomers for attachment to a“gene chip” or other support, including for examination of expressionpatterns; to raise anti-protein antibodies using DNA immunizationtechniques; and as an antigen to raise anti-DNA antibodies or elicitanother immune response. Where the polynucleotide encodes a proteinwhich binds or potentially binds to another protein (such as, forexample, in a receptor-ligand interaction), the polynucleotide can alsobe used in interaction trap assays (such as, for example, that describedin Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotidesencoding the other protein with which binding occurs or to identifyinhibitors of the binding interaction.

The proteins provided by the present invention can similarly be used inassay to determine biological activity, including in a panel of multipleproteins for high-throughput screening; to raise antibodies or to elicitanother immune response; as a reagent (including the labeled reagent) inassays designed to quantitatively determine levels of the protein (orits receptor) in biological fluids; as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state); and, of course, to isolate correlative receptors orligands. Where the protein binds or potentially binds to another protein(such as, for example, in a receptor-ligand interaction), the proteincan be used to identify the other protein with which binding occurs orto identify inhibitors of the binding interaction. Proteins involved inthese binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction.

Any or all of these research utilities are capable of being developedinto reagent grade or kit format for commercialization as researchproducts.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include withoutlimitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold SpringHarbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatiseds., 1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

6.7.2. Nutritional Uses

Polynucleotides and proteins of the present invention can also be usedas nutritional sources or supplements. Such uses include withoutlimitation use as a protein or amino acid supplement, use as a carbonsource, use as a nitrogen source and use as a source of carbohydrate. Insuch cases the protein or polynucleotide of the invention can be addedto the feed of a particular organism or can be administered as aseparate solid or liquid preparation, such as in the form of powder,pills, solutions, suspensions or capsules. In the case ofmicroorganisms, the protein or polynucleotide of the invention can beadded to the medium in or on which the microorganism is cultured.

6.7.3. Cytokine and Cell Proliferation/Differentiation Activity

A protein of the present invention may exhibit cytokine, cellproliferation (either inducing or inhibiting) or cell differentiation(either inducing or inhibiting) activity or may induce production ofother-cytokines in certain cell populations. A polynucleotide of theinvention can encode a polypeptide exhibiting such attributes. Manyprotein factors discovered to date, including all known cytokines, haveexhibited activity in one or more factor-dependent cell proliferationassays, and hence the assays serve as a convenient confirmation ofcytokine activity. The activity of a protein of the present invention isevidenced by any one of a number of routine factor dependent cellproliferation assays for cell lines including, without limitation, 32D,DA2, DAIG, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123,T1165, HT2, CTLL2, TF-1, Mo7e and CMK. The activity of a protein of theinvention may, among other means, be measured by the following methods:

Assays for T-cell or thymocyte proliferation include without limitationthose described in: Current Protocols in Immunology, Ed by J. E.Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,Pub. Greene Publishing Associates and Wiley-lnterscience (Chapter 3, InVitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7,Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolliet al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., I.Immunol. 149:3778-3783, 1992; Bowman et al., 1. Immunol. 152:1756-1761,1994.

Assays for cytokine production and/or proliferation of spleen cells,lymph node cells or thymocytes include, without limitation, thosedescribed in: Polyclonal T cell stimulation, Kruisbeek, A. M. andShevach, E. M. In Current Protocols in lmmunology. J. E. e.a. Coliganeds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; andMeasurement of mouse and human interleukin .gamma., Schreiber, R. D. InCurrent Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp.6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.

Assays for proliferation and differentiation of hematopoietic andlymphopoietic cells include, without limitation, those described in:Measurement of Human and Murine Interleukin 2 and Interleukin 4,Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols inImmunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wileyand Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211,1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc.Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse andhuman interleukin 6—Nordan, R. In Current Protocols in Immunology. J. E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto.1991; Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861, 1986;Measurement of human Interleukin 11—Bennett, F., Giannotti, J., Clark,S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. e.a.Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991;Measurement of mouse and human Interleukin 9—Ciarletta, A., Giannotti,J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology.J. E. e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto.1991.

Assays for T-cell clone responses to antigens (which will identify,among others, proteins that affect APC-T cell interactions as well asdirect T-cell effects by measuring proliferation and cytokineproduction) include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction; Chapter 6, Cytokines and their cellular receptors; Chapter 7,Immunologic studies in Humans); Weinberger et at., Proc. Natl. Acad.Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun.11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takaiet al., J. Immunol. 140:508-512, 1988.

6.7.4. Immune Stimulating or Suppressing Activity

A protein of the present invention may also exhibit immune stimulatingor immune suppressing activity, including without limitation theactivities for which assays are described herein. A polynucleotide ofthe invention can encode a polypeptide exhibiting such activities. Aprotein may be useful in the treatment of various immune deficienciesand disorders (including severe combined immunodeficiency (SCID)), e.g.,in regulating (up or down) growth and proliferation of T and/or Blymphocytes, as well as effecting the cytolytic activity of NK cells andother cell populations. These immune deficiencies may be genetic or becaused by viral (e.g., HIV) as well as bacterial or fungal infections,or may result from autoimmune disorders. More specifically, infectiousdiseases causes by viral, bacterial, fungal or other infection may betreatable using a protein of the present invention, including infectionsby HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmaniaspp., malaria spp. and various fungal infections such as candidiasis. Ofcourse, in this regard, proteins of the present invention may also beuseful where a boost to the immune system generally may be desirable,i.e., in the treatment of cancer. IL-1 has been indicated to promotetumor cell growth in cancers of various organs including breastadenocarcinoma, brain tumors, melanoma, myeloma, giant cell tumors ofbone, acute myelogenous leukemia, oral epidermoid carcinoma and squamouscell carcinoma; thus, treatment of such cancer disease states involvingelevated levels of IL-1 with IL-1 Hy1 polypeptides of the presentinvention is expected to ameliorate signs and symptoms of cancer.

Autoimmune disorders which may be treated using a protein of the presentinvention include, for example, connective tissue disease, multiplesclerosis, systemic lupus erythematosus, rheumatoid arthritis,autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmunethyroiditis, insulin dependent diabetes mellitis, myasthenia gravis,graft-versus-host disease and autoimmune inflammatory eye disease. Sucha protein (or antagonists thereof, including antibodies) of the presentinvention may also to be useful in the treatment of allergic reactionsand conditions (e.g., anaphylaxis, serum sickness, drug reactions, foodallergies, insect venom allergies, mastocytosis, allergic rhinitis,hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopicdermatitis, allergic contact dermatitis, erythema multiforme,Stevens-Johnson syndrome, allergic conjunctivitis, atopickeratoconjunctivitis, venereal keratoconjunctivitis, giant papillaryconjunctivitis and contact allergies), such as asthma (particularlyallergic asthma) or bronchitis (including chronic bronchitis) and otherrespiratory problems. Other conditions, in which immune suppression isdesired (including, for example, organ transplantation), may also betreatable using a protein (or antagonists thereof) of the presentinvention. The therapeutic effects of IL-1 Hy1 polypeptides orantagonists thereof on allergic reactions can be evaluated by in vivoanimals models such as the cumulative contact enhancement test (Lastbomet al., Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al.,Allergy 54: 446-54, 1999), guinea pig skin sensitization test (Vohr etal., Arch. Toxocol. 73: 501-9), and murine local lymph node assay(Kimber et al., J. Toxicol. Environ. Health 53: 563-79).

Using the proteins of the invention it may also be possible to modulateimmune responses, in a number of ways. Down regulation may be in theform of inhibiting or blocking an immune response already in progress ormay involve preventing the induction of an immune response. Thefunctions of activated T cells may be inhibited by suppressing T cellresponses or by inducing specific tolerance in T cells, or both.Immunosuppression of T cell responses is generally an active,non-antigen-specific, process which requires continuous exposure of theT cells to the suppressive agent. Tolerance, which involves inducingnon-responsiveness or anergy in T cells, is distinguishable fromimmunosuppression in that it is generally antigen-specific and persistsafter exposure to the tolerizing agent has ceased. Operationally,tolerance can be demonstrated by the lack of a T cell response uponreexposure to specific antigen in the absence of the tolerizing agent.

Down regulating or preventing one or more antigen functions (includingwithout limitation B lymphocyte antigen functions (such as, for example,B7)), e.g., preventing high level lymphokine synthesis by activated Tcells, will be useful in situations of tissue, skin and organtransplantation and in graft-versus-host disease (GVHD). For example,blockage of T cell function should result in reduced tissue destructionin tissue transplantation. Typically, in tissue transplants, rejectionof the transplant is initiated through its recognition as foreign by Tcells, followed by an immune reaction that destroys the transplant. Theadministration of a therapeutic composition of the invention may preventcytokine synthesis by immune cells, such as T cells, and thus acts as animmunosuppressant. Moreover, a lack of costimulation may also besufficient to anergize the T cells, thereby inducing tolerance in asubject. Induction of long-term tolerance by B lymphocyteantigen-blocking reagents may avoid the necessity of repeatedadministration of these blocking reagents. To achieve sufficientimmunosuppression or tolerance in a subject, it may also be necessary toblock the function of a combination of B lymphocyte antigens.

The efficacy of particular therapeutic compositions in preventing organtransplant rejection or GVHD can be assessed using animal models thatare predictive of efficacy in humans. Examples of appropriate systemswhich can be used include allogeneic cardiac grafts in rats andxenogeneic pancreatic islet cell grafts in mice, both of which have beenused to examine the immunosuppressive effects of CTLA4Ig fusion proteinsin vivo as described in Lenschow et al., Science 257:789-792 (1992) andTurka et al., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992). Inaddition, murine models of GVHD (see Paul ed., Fundamental Immunology,Raven Press, New York, 1989, pp. 846-847) can be used to determine theeffect of therapeutic compositions of the invention on the developmentof that disease.

Blocking antigen function may also be therapeutically useful fortreating autoimmune diseases. Many autoimmune disorders are the resultof inappropriate activation of T cells that are reactive against selftissue and which promote the production of cytokines and autoantibodiesinvolved in the pathology of the diseases. Preventing the activation ofautoreactive T cells may reduce or eliminate disease symptoms.Administration of reagents which block stimulation of T cells can beused to inhibit T cell activation and prevent production ofautoantibodies or T cell-derived cytokines which may be involved in thedisease process. Additionally, blocking reagents may induceantigen-specific tolerance of autoreactive T cells which could lead tolong-term relief from the disease. The efficacy of blocking reagents inpreventing or alleviating autoimmune disorders can be determined using anumber of well-characterized animal models of human autoimmune diseases.Examples include murine experimental autoimmune encephalitis, systemiclupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murineautoimmune collagen arthritis, diabetes mellitus in NOD mice and BBrats, and murine experimental myasthenia gravis (see Paul ed.,Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

Upregulation of an antigen function (e.g., a B lymphocyte antigenfunction), as a means of up regulating immune responses, may also beuseful in therapy. Upregulation of immune responses may be in the formof enhancing an existing immune response or eliciting an initial immuneresponse. For example, enhancing an immune response may be useful incases of viral infection, including systemic viral diseases such asinfluenza, the common cold, and encephalitis.

Alternatively, anti-viral immune responses may be enhanced in aninfected patient by removing T cells from the patient, costimulating theT cells in vitro with viral antigen-pulsed APCs either expressing apeptide of the present invention or together with a stimulatory form ofa soluble peptide of the present invention and reintroducing the invitro activated T cells into the patient. Another method of enhancinganti-viral immune responses would be to isolate infected cells from apatient, transfect them with a nucleic acid encoding a protein of thepresent invention as described herein such that the cells express all ora portion of the protein on their surface, and reintroduce thetransfected cells into the patient. The infected cells would now becapable of delivering a costimulatory signal to, and thereby activate, Tcells in vivo.

A peptide of the present invention may provide the necessary stimulationsignal to T cells to induce a T cell mediated immune response againstthe transfected tumor cells. In addition, tumor cells which lack MHCclass I or MHC class II molecules, or which fail to reexpress sufficientmounts of MHC class I or MHC class II molecules, can be transfected withnucleic acid encoding all or a portion of (e.g., a cytoplasmic-domaintruncated portion) of an MHC class I α chain protein and β₂microglobulin protein or an MHC class II α chain protein and an MHCclass II β chain protein to thereby express MHC class I or MHC class IIproteins on the cell surface. Expression of the appropriate class I orclass II MHC in conjunction with a peptide having the activity of a Blymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediatedimmune response against the transfected tumor cell. Optionally, a geneencoding an antisense construct which blocks expression of an MHC classII associated protein, such as the invariant chain, can also becotransfected with a DNA encoding a peptide having the activity of a Blymphocyte antigen to promote presentation of tumor associated antigensand induce tumor specific immunity. Thus, the induction of a T cellmediated immune response in a human subject may be sufficient toovercome tumor-specific tolerance in the subject.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for thymocyte or splenocyte cytotoxicity include,without limitation, those described in: Current Protocols in Immunology,Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience(Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19;Chapter.7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol.128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985;Takai et al., I. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982;Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol.137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992-1998; Takai etal., J. Immunol. 140:508-512, 1988; Bertagnolli et al., CellularImmunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092,1994.

Assays for T-cell-dependent immunoglobulin responses and isotypeswitching (which will identify, among others, proteins that modulateT-cell dependent antibody responses and that affect Th1/Th2 profiles)include, without limitation, those described in: Maliszewski, J.Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitroantibody production, Mond, J. J. and Brunswick, M. In Current Protocolsin Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, JohnWiley and Sons, Toronto. 1994.

Mixed lymphocyte reaction (MLR) assays (which will identify, amongothers, proteins that generate predominantly Thl and CTL responses)include, without limitation, those described in: Current Protocols inImmunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M.Shevach, W. Strober, Pub. Greene Publishing Associates andWiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai etal., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

Dendritic cell-dependent assays (which will identify, among others,proteins expressed by dendritic cells that activate naive T-cells)include, without limitation, those described in: Guery et al., J.Immunol. 134:536-544, 1995; Inaba et al., Journal of ExperimentalMedicine 173:549-559, 1991; Macatonia et al., Journal of Immunology154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993;Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal ofExperimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal ofClinical Investigation 94:797-807, 1994; and Inaba et al., Journal ofExperimental Medicine 172:631-640, 1990.

Assays for lymphocyte survival/apoptosis (which will identify, amongothers, proteins that prevent apoptosis after superantigen induction andproteins that regulate lymphocyte homeostasis) include, withoutlimitation, those described in: Darzynkiewicz et al., Cytometry13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca etal., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243,1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai etal., Cytometry 14:891-897, 1993; Gorczyca et al., International Journalof Oncology 1:639-648, 1992.

Assays for proteins that influence early steps of T-cell commitment anddevelopment include, without limitation, those described in: Antica etal., Blood 84:111-117, 1994; Fine et al., Cellular Immunology155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al.,Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.

6.7.5. Hematopoiesis Regulating Activity

A protein of the present invention may be useful in regulation ofhematopoiesis and, consequently, in the treatment of myeloid or lymphoidcell deficiencies. Even marginal biological activity in support ofcolony forming cells or of factor-dependent cell lines indicatesinvolvement in regulating hematopoiesis, e.g. in supporting the growthand proliferation of erythroid progenitor cells alone or in combinationwith other cytokines, thereby indicating utility, for example, intreating various anemias or for use in conjunction withirradiation/chemotherapy to stimulate the production of erythroidprecursors and/or erythroid cells; in supporting the growth andproliferation of myeloid cells such as granulocytes andmonocytes/macrophages (i.e., traditional CSF activity) useful, forexample, in conjunction with chemotherapy to prevent or treat consequentmyelo-suppression; in supporting the growth and proliferation ofmegakaryocytes and consequently of platelets thereby allowing preventionor treatment of various platelet disorders such as thrombocytopenia, andgenerally for use in place of or complimentary to platelet transfusions;and/or in supporting the growth and proliferation of hematopoietic stemcells which are capable of maturing to any and all of theabove-mentioned hematopoietic cells and therefore find therapeuticutility in various stem cell disorders (such as those usually treatedwith transplantation, including, without limitation, aplastic anemia andparoxysmal nocturnal hemoglobinuria), as well as in repopulating thestem cell compartment post irradiation/chemotherapy, either in-vivo orex-vivo (i.e., in conjunction with bone marrow transplantation or withperipheral progenitor cell transplantation (homologous or heterologous))as normal cells or genetically manipulated for gene therapy.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for proliferation and differentiation of varioushematopoietic lines are cited above.

Assays for embryonic stem cell differentiation (which will identify,among others, proteins that influence embryonic differentiationhematopoiesis) include, without limitation, those described in:Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al.,Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al.,Blood 81:2903-2915, 1993.

Assays for stem cell survival and differentiation (which will identify,among others, proteins that regulate lympho-hematopoiesis) include,without limitation, those described in: Methylcellulose colony formingassays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y.1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;Primitive hematopoietic colony forming cells with high proliferativepotential, McNiece, I. K. and Briddell, R. A. In Culture ofHematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39,Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., ExperimentalHematology 22:353-359, 1994; Cobblestone area forming cell assay,Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I. Freshney, etal. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long termbone marrow cultures in the presence of stromal cells, Spooncer, E.,Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y.1994; Long term culture initiating cell assay, Sutherland, H. J. InCulture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp.139-162, Wiley-Liss, Inc., New York, N.Y. 1994.

6.7.6. Tissue Growth Activity

A protein of the present invention also may have utility in compositionsused for bone, cartilage, tendon, ligament and/or nerve tissue growth orregeneration, as well as for wound healing and tissue repair andreplacement, and in the treatment of burns, incisions and ulcers.

A protein of the present invention, which induces cartilage and/or bonegrowth in circumstances where bone is not normally formed, hasapplication in the healing of bone fractures and cartilage damage ordefects in humans and other animals. Such a preparation employing aprotein of the invention may have prophylactic use in closed as well asopen fracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery.

A protein of this invention may also be used in the treatment ofperiodontal disease, and in other tooth repair processes. Such agentsmay provide an environment to attract bone-forming cells, stimulategrowth of bone-forming cells or induce differentiation of progenitors ofbone-forming cells. A protein of the invention may also be useful in thetreatment of osteoporosis or osteoarthritis, such as through stimulationof bone and/or cartilage repair or by blocking inflammation or processesof tissue destruction (collagenase activity, osteoclast activity, etc.)mediated by inflammatory processes.

Another category of tissue regeneration activity that may beattributable to the protein of the present invention is tendon/ligamentformation. A protein of the present invention, which inducestendon/ligament-like tissue or other tissue formation in circumstanceswhere such tissue is not normally formed, has application in the healingof tendon or ligament tears, deformities and other tendon or ligamentdefects in humans and other animals. Such a preparation employing atendon/ligament-like tissue inducing protein may have prophylactic usein preventing damage to tendon or ligament tissue, as well as use in theimproved fixation of tendon or ligament to bone or other tissues, and inrepairing defects to tendon or ligament tissue. De novotendon/ligament-like tissue formation induced by a composition of thepresent invention contributes to the repair of congenital, traumainduced, or other tendon or ligament defects of other origin, and isalso useful in cosmetic plastic surgery for attachment or repair oftendons or ligaments. The compositions of the present invention mayprovide environment to attract tendon- or ligament-forming cells,stimulate growth of tendon- or ligament-forming cells, inducedifferentiation of progenitors of tendon- or ligament-forming cells, orinduce growth of tendon/ligament cells or progenitors ex vivo for returnin vivo to effect tissue repair. The compositions of the invention mayalso be useful in the treatment of tendinitis, carpal tunnel syndromeand other tendon or ligament defects. The compositions may also includean appropriate matrix and/or sequestering agent as a carrier as is wellknown in the art.

The protein of the present invention may also be useful forproliferation of neural cells and for regeneration of nerve and braintissue, i.e. for the treatment of central and peripheral nervous systemdiseases and neuropathies, as well as mechanical and traumaticdisorders, which involve degeneration, death or trauma to neural cellsor nerve tissue. More specifically, a protein may be used in thetreatment of diseases of the peripheral nervous system, such asperipheral nerve injuries, peripheral neuropathy and localizedneuropathies, and central nervous system diseases, such as Alzheimer's,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome. Further conditions which may betreated in accordance with the present invention include mechanical andtraumatic disorders, such as spinal cord disorders, head trauma andcerebrovascular diseases such as stroke. Peripheral neuropathiesresulting from chemotherapy or other medical therapies may also betreatable using a protein of the invention.

Proteins of the invention may also be useful to promote better or fasterclosure of non-healing wounds, including without limitation pressureulcers, ulcers associated with vascular insufficiency, surgical andtraumatic wounds, and the like.

It is expected that a protein of the present invention may also exhibitactivity for generation or regeneration of other tissues, such as organs(including, for example, pancreas, liver, intestine, kidney, skin,endothelium), muscle (smooth, skeletal or cardiac) and vascular(including vascular endothelium) tissue, or for promoting the growth ofcells comprising such tissues. Part of the desired effects may be byinhibition or modulation of fibrotic scarring to allow normal tissue toregenerate. A protein of the invention may also exhibit angiogenicactivity.

A protein of the present invention may also be useful for gut protectionor regeneration and treatment of lung or liver fibrosis, reperfusioninjury in various tissues, and conditions resulting from systemiccytokine damage.

A protein of the present invention may also be useful for promoting orinhibiting differentiation of tissues described above from precursortissues or cells; or for inhibiting the growth of tissues describedabove.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assays for tissue generation activity include, without limitation, thosedescribed in: International Patent Publication No. WO95/16035 (bone,cartilage, tendon); International Patent Publication No. WO95/05846(nerve, neuronal); International Patent Publication No. WO91/07491(skin, endothelium).

Assays for wound healing activity include, without limitation, thosedescribed in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, H.I. and Rovee, D. T., eds.), Year Book Medical Publishers, Inc., Chicago,as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84(1978).

6.7.7. Axtivin/Inhibin Activity

A protein of the present invention may also exhibit activin- orinhibin-related activities. A polynucleotide of the invention may encodea polypeptide exhibiting such characteristics. Inhibins arecharacterized by their ability to inhibit the release of folliclestimulating hormone (FSH), while activins and are characterized by theirability to stimulate the release of follicle stimulating hormone (FSH).Thus, a protein of the present invention, alone or in heterodimers witha member of the inhibin α-family, may be useful as a contraceptive basedon the ability of inhibins to decrease fertility in female mammals anddecrease spermatogenesis in male mammals. Administration of sufficientamounts of other inhibins can induce infertility in these mammals.Alternatively, the protein of the invention, as a homodimer or as aheterodimer with other protein subunits of the inhibin-β group, may beuseful as a fertility inducing therapeutic, based upon the ability ofactivin molecules in stimulating FSH release from cells of the anteriorpituitary. See, for example, U.S. Pat. No. 4,798,885. A protein of theinvention may also be useful for advancement of the onset of fertilityin sexually immature mammals, so as to increase the lifetimereproductive performance of domestic animals such as cows, sheep andpigs.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assays for activin/inhibin activity include, without limitation, thosedescribed in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al.,Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Masonet al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci.USA 83:3091-3095, 1986.

6.7.8. Chemotactic/Chemokinetic Activity

A protein of the present invention may have chemotactic or chemokineticactivity (e.g., act as a chemokine) for mammalian cells, including, forexample, monocytes, fibroblasts, neutrophils, T-cells, mast cells,eosinophils, epithelial and/or endothelial cells. A polynucleotide ofthe invention can encode a polypeptide exhibiting such attributes.Chemotactic and chemokinetic proteins can be used to mobilize or attracta desired cell population to a desired site of action. Chemotactic orchemokinetic proteins provide particular advantages in treatment ofwounds and other trauma to tissues, as well as in treatment of localizedinfections. For example, attraction of lymphocytes, monocytes orneutrophils to tumors or sites of infection may result in improvedimmune responses against the tumor or infecting agent.

A protein or peptide has chemotactic activity for a particular cellpopulation if it can stimulate, directly or indirectly, the directedorientation or movement of such cell population. Preferably, the proteinor peptide has the ability to directly stimulate directed movement ofcells. Whether a particular protein has chemotactic activity for apopulation of cells can be readily determined by employing such proteinor peptide in any known assay for cell chemotaxis.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assays for chemotactic activity (which will identify proteins thatinduce or prevent chemotaxis) consist of assays that measure the abilityof a protein to induce the migration of cells across a membrane as wellas the ability of a protein to induce the adhesion of one cellpopulation to another cell population. Suitable assays for movement andadhesion include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Marguiles, E. M. Shevach, W. Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 6.12, Measurement of alpha and betaChemokines 6.12.1-6.12.28); Taub et al. J. Clin. Invest. 95:1370-1376,1995; Lind et al. APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol.25:1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnstonet al. J. of Immunol. 153:1762-1768, 1994.

6.7.9. Hemostatic and Thrombolytic Activity

A protein of the invention may also exhibit hemostatic or thrombolyticactivity. A polynucleotide of the invention can encode a polypeptideexhibiting such attributes. Such a protein is expected to be useful intreatment of various coagulation disorders (including hereditarydisorders, such as hemophilias) or to enhance coagulation and otherhemostatic events in treating wounds resulting from trauma, surgery orother causes. A protein of the invention may also be useful fordissolving or inhibiting formation of thromboses and for treatment andprevention of conditions resulting therefrom (such as, for example,infarction of cardiac and central nervous system vessels (e.g., stroke).

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assay for hemostatic and thrombolytic activity include, withoutlimitation, those described in: Linet et al., J. Clin. Pharmacol.26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987;Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins35:467-474, 1988.

6.7.10. Receptor/Ligand Activity

A protein of the present invention may also demonstrate activity asreceptors, receptor ligands or inhibitors or agonists of receptor/ligandinteractions. A polynucleotide of the invention can encode a polypeptideexhibiting such characteristics. Examples of such receptors and ligandsinclude, without limitation, cytokine receptors and their ligands,receptor kinases and their ligands, receptor phosphatases and theirligands, receptors involved in cell-cell interactions and their ligands(including without limitation, cellular adhesion molecules (such asselectins, integrins and their ligands) and receptor/ligand pairsinvolved in antigen presentation, antigen recognition and development ofcellular and humoral immune responses). Receptors and ligands are alsouseful for screening of potential peptide or small molecule inhibitorsof the relevant receptor/ligand interaction. A protein of the presentinvention (including, without limitation, fragments of receptors andligands) may themselves be useful as inhibitors of receptor/ligandinteractions.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for receptor-ligand activity include without limitationthose described in: Current Protocols in Immunology, Ed by J. E.Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28,Measurement of Cellular Adhesion under static. conditions7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868,1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein etal., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol.Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.

By way of example, the novel Interleukin-1 Receptor Antagonist of theinvention may be used as a ligand for a cytokine receptor therebymodulating the biological activity of that receptor. Examples ofcytokine receptors that may be used include, but are not limited to, theInterleukin-1 Type I or the Interleukin-Type II Receptor. Whether thenovel Interleukin-1 Receptor Antagonist of the invention exhibitsagonist, partial agonist, antagonist, or partial antagonist activity fora particular receptor, such as a cytokine receptor, in a particular celltype can be determined by conventional techniques known to those skilledin the art. In one embodiment, one or more cells expressing a cytokinereceptor (e.g., Interleukin-1 Type I or Type II Receptors) are contactedwith the protein of the invention. Examples of cells that may becontacted with the protein of the invention include, but are not limitedto, mammalian cells such as fibroblasts and T-cells. Preferably thenovel protein of the invention acts as an antagonist for a cytokinereceptor (e.g.-the Interleukin-I Receptor) so that the biologicalactivities of that receptor are inhibited.

Studies characterizing drugs or proteins as agonist or antagonist orpartial agonists a partial antagonist require the use of other proteinsas competing ligands. The novel protein of the present invention exhibitan affinity for Interleukin-1 Receptor. Thus, the proteins of thepresent invention may be used, for example, as competitors in assaysinvolving Interleukin-1 Receptors (e.g., Example 12). Alternatively ,theprotein of the invention may be labeled by being coupled toradioisotopes, colorimetric molecules or a toxin molecules byconventional methods. (“Guide to Protein Purification” Murray P.Deutscher (ed) Methods in Enzymology Vol. 182 (1990) Academic Press,Inc. San Diego) and used in both in vivo and in vitro to bind to theInterleukin-1 Receptor. Examples of radioisotopes include, but are notlimited to, tritium and carbon-14 . Examples of colorimetric moleculesinclude, but are not limited to, fluorescent molecules such asfluorescamine, or rhodamine or other calorimetric molecules. Examples oftoxins include, but are not limited, to riacin. By way of example, theproteins coupled to such molecules are useful in studies involving invivo or in vitro metabolism of the Interleukin-1 Receptor.

6.7.11 Drug Screening with the Novel Interleukin-1 Receptor AntagonistPolypeptides

This invention is particularly useful for screening compounds by usingthe novel Interleukin-1 Receptor Antagonist polypeptides or bindingfragments thereof in any of a variety of drug screening techniques. Thenovel Interleukin-1 receptor antagonist polypeptides or fragmentsemployed in such a test may either be free in solution, affixed to asolid support, borne on a cell surface or located intracellularly. Onemethod of drug screening utilizes eukaryotic or prokaryotic host cellswhich are stably transformed with recombinant nucleic acids expressingthe polypeptide or fragment. Drugs are screened against such transformedcells in competitive binding assays. Such cells, either in viable orfixed form, can be used for standard binding assays. One may measure,for example, the formation of complexes between novel Interleukin-1receptor antagonist polypeptides or fragments and the agent being testedor examine the diminution in complex formation between the novelInterleukin-1 receptor antagonist polypeptides and an appropriate cellline, which are well known in the art.

6.7.11.1 Assay for Anti-Interleukin-1 Receptor Activity

In one embodiment, the Interleukin-1 receptor antagonist activity of thepolypeptides of the invention is determined using a method that involve(1) forming a mixture comprising Interleukin-1, the Interleukin-1receptor, and the Interleukin-1 receptor antagonist polypeptides of theinvention and/or its agonists and antagonists (or agonist or antagonistdrug candidates) and/or antibodies specific for the Interleukin-1receptor antagonist polypeptides of the invention; (2) incubating themixture under conditions whereby, but for the presence of saidInterleukin-1 receptor antagonist polypeptide of the invention and/orits agonists and antagonists (or agonist or antagonist drug candidates)and/or antibodies specific for the Interleukin-1 receptor antagonistpolypeptides of the invention, the Interleukin-1 binds to theInterleukin-1 receptor; and (3) detecting the presence or absence ofspecific binding of Interleukin-1 to the Interleukin-1 receptor.

6.7.11.2 Assay for Antagonists and Agonists

Human HepG2 cells are incubated at 37 degree(s) C. for 18-24 hours inserum-free Dulbecco's modified Eagle medium. Separate monolayers ofcells are incubated in the same medium supplemented with Interleukin-1at various concentrations and in the same medium supplemented withInterleukin-1 receptor antagonists of the invention at variousconcentrations.

Monolayers are rinsed vigorously with isotonic buffer and incubated in(35-S) methionine, 250 mu ci/ml methionine-free medium and pulsed for aperiod of 15-30 minutes to assess net synthesis. Cell culture fluid isdiscarded and monolayers are again rinsed and resuspended in cell lysisbuffer. The newly synthesized radiolabeled hepatic proteins in thesecell lysates are detected by immunoprecipitation, SDS-PAGE andfluorography.

6.7.12. Anti-Inflammatory Activity

Proteins of the present invention may also exhibit anti-inflammatoryactivity. The anti-inflammatory activity may be achieved by providing astimulus to cells involved in the inflammatory response, by inhibitingor promoting cell-cell interactions (such as, for example, celladhesion), by inhibiting or promoting chemotaxis of cells involved inthe inflammatory process, inhibiting or promoting cell extravasation, orby stimulating or suppressing production of other factors which moredirectly inhibit or promote an inflammatory response. Proteinsexhibiting such activities can be used to treat inflammatory conditionsincluding chronic or acute conditions), including without limitationintimation associated with infection (such as septic shock, sepsis orsystemic inflammatory response syndrome (SIRS)), ischemia-reperfusioninjury, endotoxin lethality, arthritis, complement-mediated hyperacuterejection, nephritis, cytokine or chemokine-induced lung injury,inflammatory bowel disease, Crohn's disease or resulting from overproduction of cytokines such as TNF or IL-1. Proteins of the inventionmay also be useful to treat anaphylaxis and hypersensitivity to anantigenic substance or material. In particular, the Interleukin-1receptor antagonist of this invention may be utilized to prevent ortreat condition such as, but not limited to, utilized, for example, aspart of methods for the prevention and/or treatment of disordersinvolving sepsis, acute pancreatitis, endotoxic shock, cytokine inducedshock, rheumatoid arthritis, chronic inflammatory arthritis, pancreaticcell damage from diabetes mellitus type 1, graft versus host disease,inflammatory bowel disease, inflammation associated with pulmonarydisease, including bronchitis and chronic bronchitis, other autoimmunedisease or inflammatory disease, including allergies and in particularchronic allergies, an antiproliferative agent such as for acute orchronic myelogenous leukemia or in the prevention of premature laborsecondary to intrauterine infections.

6.7.12.1. Regulation of IL-13 Anti-Inflammatory Activity

IL1-Hy1 proteins of the invention can induce expression of theanti-inflammatory cytokine IL-13. As a result, IL-1 Hy1 polynucleotidesor polypeptides, or antagonists of IL-1 Hy1 activity can be used fortherapeutic intervention in any of the numerous pathological states inwhich IL-13 activity has been implicated.

For example, recent animal model data suggest that IL-13 is a centralcytokine in promoting atopic asthma [Ahmed et al., Exp Clin Immunogenet17(1):18-22 (2000)], through the stimulation of bronchial epithelialmucus secretion and smooth muscle hyper-reactivity [Heinzmann et al.,HumMol Genet 9(4):549-59 (2000)]. Certain variants of IL-13 signaling arelikely to be important promoters of human asthma [Heinzmann et al.,HumMol Genet 9(4):549-59 (2000)]. IL-13, which shares a receptor componentand signaling pathways with IL-4, was found to be necessary andsufficient for the expression of allergic asthma in a manner that isindependent of immunoglobulin E and cosinophils. [Wills-Karp et al.,Science 282(5397):2258-61 (1998)]. Inhibition of IL-13 activity throughadministration of IL-1Hy1 antagonists is thus expected to reduce theadverse effects of asthma.

Other data, however, indicates that IL-13 (but not human IL-4) exhibitsan anti-inflammatory action in the airways of TNF-alpha- orantigen-challenged guinea pigs, by mechanisms that may involve thedecreased generation of eosinophil-stimulating activity in the airways[Watson et al., J.Respir. Cell Mol Biol 20(5):1007-12 (1999)]. IL-13 mayplay a role in the pathogenesis of both atopic and nonatopic asthma, atleast partly through promoting recruitment of eosinophils to thebronchial mucosa [Humbert et al.,J Allergy Clin Immunol 99(5):657-65(1997)].

Administration of IL-13 has been suggested as a potential therapeuticapproach in the prevention of type 1 diabetes [Kretowski et al, Scand JImmunol 51(3):321-5 (2000)]. IL-13 treatment on development of type 1diabetes in diabetes-prone nonobese diabetic (NOD) mice indicated thathIL-13 is capable of downregulating immunoinflammatory diabetogenicpathways in NOD mice [Zaccone et al, Diabetes 48(8):1522-8 (1999)].Administration of IL-Hy1 polynucleotides, polypeptides and agonists istherefore expected to be useful for preventing or treating type Idiabetes.

Recent evidence suggests a role for IL-12 and IL-13 in modulatingallergic responses and support the notion that the clinical effects ofglucocorticoids are at least partially mediated through the modulationof cytokine production [Naseer et al., Am J Respir Crit Care Med155(3):845-51(1997)]. Additional evidence indicates a distinct role forIL-13 in mediating the physiologic response to a diverse array ofallergens and parasites [Corry, Curr Opin Immunol 11(6):610-4 (1999)].For example, IL-13 has been shown to be a key factor in determiningsusceptibility to Leishmania major infection as indicated by datashowing that IL-13 in transgenic mice makes the normally resistantC57BL/6 mouse strain susceptible to L. major infection even in theabsence of IL-4 expression [Matthews et al., J Immunol 164(3):1458-62(2000)]. Similarly, the role of IL-13 in helminth-induced inflammationand protective immunity against nematode infections was demonstrated inrecent observations which indicated that IL-13 can have equal or evengreater importance than IL-4 in inflammatory responses and hostprotection against infection [Finkelman et al., Curr Opin Immunol Aug.11, 1999 (4):420-6 ]. Also, data suggests a role for IL-13 in resistanceto intestinal nematode infection [Bancroft et al, J Immunol160(7):3453-61 (1998)]. Still other data suggest that IL-13 expressionis a prominent feature of the allergen-induced late nasal response(LNR), and that inhibition of the LNR following steroid therapy may bepartly attributable to inhibition of IL-13 expression. [Ghaffar etal.,Am J Respir Celi Mol Biol 17(l):17-24 (1997)]. Increased IL-13expression is also an important feature of allergic and nonallergicpatients with chronic sinusitis [al Ghamdi et al., J Otolaryngol26(3):160-6 (1997)]. IL-13 may play some role in the development ofatopic dernatitis (AD) [Takamatsu et al., Dermatology 196(4):377-81(1998)]. Levels of mRNA for IL-13 were significantly greater in PBMC ofpatients with AD than in controls, suggesting that an increase in IL-13expression may regulate the in vivo synthesis of IgE in patients withAD. [Katagiri et al., Clin Exp Immunol 108(2):289-94 (1997)].

IL-13 also provides protection from LPS-induced lethal endotoxemia in amanner that is similar to but independent from that of IL-10, andtherefore can be added to the list of cytokine immunomodulators thatmight be beneficial in the treatment of septic shock. [Muchamuel et al.,J Immunol 158(6):2898-903 (1997)]. IL-13 (0.5 microgram/mouse)dramatically reduced the lethal effects of lipopolysaccharide (LPS) ifadministered either 24 or 4 h prior to or concomitantly with LPSchallenge [Nicoletti et al., Eur J Immunol 27(6):1580-3 (1997)].Administration of IL-1Hy1 polynucleotides, polypeptides or otheragonists is thus expected to protect against the adverse effects ofendotoxemia.

IL-13 has also been reported to suppress various aspects of thepathophysiology of arthritis. [Evans et al., Intern Med 38(3):233-9(1999)], and administration of IL-1Hy1 polynucleotides, polypeptides orother agonists is thus expected to be useful for treating arthritis.

Involvement of IL-13 has also been implicated is numerous otherpathological states. For example, data suggests a role for interleukin13 in Hodgkin's disease [Fricker, Mol Med Today 5(11):463 (1999)]. IL-13activates STAT6 and inhibits liver injury induced byischemia/reperfusion. IL-13 suppresses liver neutrophil recruitment,hepatocellular injury, and liver edema were each reduced [Yoshidome etal., Am J Pathol 155(4):1059-64 (1999)]. Administration of IL-13 appearsto be promising for treatment for severe uveitis [Roberge et al., Br JOphthalmol 82(10):1195-8 (1998)]. An interleukin 13 (IL-13) receptor hasalso be observed to be overexpressed in several AIDS-KS cell linesexamined. [Husain et la., Clin Cancer Res 3(2):151-6 (1997)]. Th2cytokines, such as IL-13, play a part in the development of Kimura'sdisease [Katagiri et al., BrJ Dermatol 137(6):972-7 (1997)]. Resultshave suggested that reduced amounts of IL-13 in the peritoneal fluid ofwomen with endometriosis may lead to a lack of suppression of macrophageactivation, thereby contributing to the overall pathogenesis of thisdisease [McLaren et al., Hum Reprod 12(6):1307-10 (1997)]. IL-13 may bea serologic indicator of systemic inflammation in patients with systemicsclerosis (SSc). [Hasegawa et al., J Rheumatol 24(2):328-32 (1997)].

6.7.12.2 Modulation of IL-18, IL-12 and IFN-γ Related Disorders

Administration of IL-1Hy1 polynucleotides, polypeptides and agonists isalso expected to be useful for the treatment of IL-18 and/or IL-12and/or IFN-γ related disorders. IL-1Hy1 inhibits IL-18 and IL-12activity, including IL-18 and IL-12 induced IFN-γ production.

IL-18 has been found to have a variety of biological activitiesincluding the stimulation of activated T cell proliferation, enhancementof NK cell lytic activity, induction of IFNγ secretion, enhancement ofFas ligand expression and function, and stimulation ofgranulocyte-macrophage colony-stimulating factor (GM-CSF) production byactivated T cells. IL-18 has been shown to counteract viral andintracellular infections and suppress tumor formation. However, IL-18 isalso involved in the pathogenic progression of chronic inflammatorydiseases, including endotoxin-induced shock, liver injury (includingendotoxin-induced liver injury, hepatitis, biliary atresia andobesity-related fatty liver) and autoimmune diseases. Other disordersrelated to IL-18 production include meliodosis, purine nucleosidephosphorylase deficiency, increased susceptibility to Leishmania majorand Staphylococcus aureus infection, hemophagocytic lymphohistiocytosis,mononucleosis, viral meningitis/encephalitis, bacterialmeningitis/encephalitis and ischemia or ischemia/reperfusion injury.

Inflammation may result from infection with pathogenic organisms(including gram-positive bacteria, gram-negative bacteria, viruses,fungi, and parasites such as protozoa and helminths), transplantrejection (including rejection of solid organs such as kidney, liver,heart, lung or cornea, as well as rejection of bone marrow transplantsincluding graft versus host disease (GVHD)), or from localized chronicor acute autoimmune or allergic reactions. Autoimmune diseases includeacute glomerulonephritis; rheumatoid or reactive arthritis; chronicglomerulonephritis; inflammatory bowel diseases such as Crohn's disease,ulcerative colitis and necrotizing enterocolitis; granulocytetransfusion associated syndromes; inflammatory dermatoses such ascontact dermatitis, atopic dermatitis, psoriasis; systemic lupuserythematosus (SLE), autoimmune thyroiditis, multiple sclerosis, someforms of diabetes, or any other autoimmune state where attack by thesubject's own immune system results in pathologic tissue destruction.Allergic reactions include allergic asthma, chronic bronchitis, allergicrhinitis, acute and delayed hypersensitivity. Systemic inflammatorydisease states include inflammation associated with trauma, burns,reperfusion following ischemic events (e.g. thrombotic events in heart,brain, intestines or peripheral vasculature, including myocardialinfarction and stroke), sepsis, ARDS or multiple organ dysfunctionsyndrome. Inflammatory cell recruitment also occurs in atheroscleroticplaques.

Endotoxin activation of the systemic inflammatory response leads to anumber of disorders including bacterial and/or endotoxin-related shock,fever, tachycardia, tachypnea, cytokine overstimulation, increasedvascular permeability, hypotension, complement activation, disseminatedintravascular coagulation, anemia, thrombocytopenia, leukopenia,pulmonary edema, adult respiratory distress syndrome, intestinalischemia, renal insufficiency and failure, and metabolic acidosis.

Hepatitis represents liver disorders that are characterized by hepaticinflammation and necrosis that can be manifested as an acute or chroniccondition. These liver disorders include virus-induced hepatitis such ashepatitis A, hepatitis B, hepatitis C (non-A, non-B hepatitis),hepatitis D, hepatitis E; toxin and drug induced hepatitis such asacetaminophohen hepatotoxicity, halothane hepatotoxicity, mehtyldopahepatoxicity, iaoniazid hepatoxicity, sodium valproate hepatoxicity,phenytion hepatoxicity, chlorpromazine hepatoxicity, amiodaronehepatoxicity, amioidarone hepatoxicity, erythromycin hepatoxicity, oralcontraceptive hepatoxicity, 17,α-alkyl-substituted anabolic steroidhepatoxicity and trimethoprim-sulfamethoxazole hepatoxicity; cholestatichepatitis; alcoholic hepatitis; autoimmune chronic active hepatitis; andT cell mediated hepatitis. Other conditions that cause liver injuryinclude congenital bilary atresia, obesity-related fatty liver and theautosomal recessive disease heamophagocytic lymphohistocytosis (HLH).

IL-18 induced IFN-γ plays a role in liver injury. IFNγ has been shown tomediate LPS-induced liver injury following Propionibacterium acnesinfection as described in Tsuji et al. (J. Immunol. 162: 1049-55, 1999).Large number of macrophages and lymphocytes infiltrate the portal areain response to P. acnes infection which results in intrahepaticformation of granulomas. IFNγ knock out mice exhibited less macrophageinfiltration and a reduction in the number and size of granulomas.Subsequent treatment with low doses of LPS caused massive hepaticnecrosis and increased IL-12, IL-18 and TNF-α serum levels in the normalmice, while the knock out mice exhibited drastic decreases in IL-12,IL-18 and TNF-α serum levels. The addition of IFNγ neutralizing antibodyalso caused a decrease in IL-18 and IL-12 levels. This model of liverinjury indicates that LPS-induced liver injury is associated withincreased levels of IL-18, IL-12 and IFN-γ. Currently, a role for IL-1βis not known in this liver injury model. Since IL-1β is known to beinduced by LPS, it is possible IL-1β also plays a role in the disorder.Treatment with IL-Ra may modulate the severity of liver injury due toIL-18 induced IFN-γ production and IL-1β.

IL-18 has also been shown to be involved in the immunomediated hepatitismodel where treatment with concavalin A induced hepatitis in mice asdescribed by Fiorucci et al. (Gastroenterology 118: 404-21, 2000). Inthis model, CD+ Tcells and Th1-like cytokines cause Fas mediated livercell death. Treatment with a nitric oxide derivative of aspirinprotected against this cell death by reducing production of IFNγ, IL-18,IL-12, IL-1β and TNF-α. In addition, a neutralizing antibody to IL-18caused a decrease in IFNγ production and reduced liver injury induced byconA.

HLH is a fatal autosomal recessive disease that manifests in earlychildhood. This disease is characterized by fever, hepatosplenomegaly,cytopenia and widespread infiltration of vital organs by activatedlymphocytes and macrophages. Patients with HLH exhibit elevated serumlevels of IL-18. IL-18 plays an important role in the induction of ThIcells in HLH patients. (Takada et al., Br. J. Haematol. 106: 182-9,1999).

IL-1Hy1 inhibits IL-18 induced production of IFNγ. In the modelsdescribed above, the degree of IL-1β activity is not known. Since IL-1βis known to be induced by LPS, it is possible that IL-1β also play arole in the pathogenicity of these conditions. The presence of theappropriate amount of IL-1Hy1 polynucleotides, polypeptides or otheragonists may modulate the severity of the disease states due to bothIL-18 induced IFNγ production and IL-1β.

IL-12 is known to potentiate IFNγ production, and the cytolytic activityof NK cells and cytotoxic T lymphocytes. These immunomodulatory effectshave implicated a role for IL-12 in therapies for cancer and infectiousdisease. However, these same therapeutic effects can also promoteautoimmune diseases and chronic inflammatory conditions suchas.:multiple sclerosis, transplant rejection and cytotoxicity.

IL-12 and IFN-γ are involved in the pathogenesis of multiple sclerosis(MS). In the experimental allergic encephalomyelitis animal model (EAE),the demyelinating effect on the central nervous system is carried outsimilar to that in humans suffering from MS. Currently, IFNβ is used totreat MS. The mechanism of IFNβ treatment may be to decrease the numberof IFNγ producing T cells in MS patients. (Rep et al., J. Neuroimmunol.96:92-100, 1999). In addition, IFNγ production in blood lymphocytes wasfound to correlate with disability score in MS patients. (Petcreit etal., Mult. Scler. 6: 19-23, 2000). Antibodies against IL-12 were foundto prevent superantigen-induced and spontaneous relapses of EAE in mice(Constantineseu et al., J. Immunol. 161: 5097-5104, 1998). All thesestudies point to the involvement of IL-12 induced IFNγ production in theprogression of MS in human patients. Therefore, treatment with IL-1Hy1polynucleotides, polypeptides or other agonists to reduce IFNγproduction may be an useful therapy for MS patients.

The combination of IL-12 and IL-2 has synergistic anti-tumor activity invivo. However, in clinical trials the combination resulted insignificant toxicity and subsequently shock and mortality. (Cohen,Science 270: 908 1995). In a murine model investigated by Carson et al.(J. Immunol., 162: 4943-5, 1999) determined that the fatal systemicinflammatory response was NK cell dependent but not related to othereffector molecules in the system such as IL-1, TNF-α, and IFNγ. IL-1Hy1polynucleotides, polypeptides or other agonists is expected to inhibitIL-12 induced IFN-γ production and is expected to inhibit otherbiological activities of IL-12 such as NK cell cytolytic activity.Inhibition of NK cell activity, through IL-Ra administration, may reducetoxicity resulting from IL-12 antitumor treatment.

The effect of IL-1Hy1 on IL-12 and/or IL-18 activity may be determinedby measuring the biological activities of these cytokines. Both IL-12and IL-18 are known to induce IFNγ production in T cells. In addition toIFN-γ, the combination of IL-12 and IL-18 increases production of IL-3,IL-6 and TNF. Treatment with IL-1Hy1 is expected to reduce IFNγproduction induced by IL-12 and IL-18. Circulating or local levels ofIFNγ in tissue or fluid samples from patients treated with IL-1Hy1polynucleotides, polypeptides or other agonists will be an indication ofthe therapeutic effects of IL-1Hy1 on the IL-18 and IL-12 relateddisorders. Tissue samples include tissue samples from an area involvedin inflammation or other disease. Fluid samples include, for example,whole blood, plasma, serum, cerebrospinal fluid, synovial fluid,peritoneal fluids (including lavage fluids or exudate), pleural fluids(including lavage fluids or exudate), wound fluids (including lavagefluids or exudate).

Furthermore, IL-12 is known to activate NK cells and to decrease serumIgE levels. These assays may also be used to measure the effectivenessof IL-1Hy1 treatment for IL-12 related disorders. The NK cell cytolyticactivity in patients treated with IL-1Hy1 polynucleotides, polypeptidesor other agonists can be assayed by measuring patient's blood samplesability to lysis colon carcinoma or lymphoma cells in vitro. (Liebermanet al., J. Sur. Res., 50: 410-415, 1992) In addition, the serum levelsof IgE from patients treated with IL-1Hy1 can be measured to determinethe effectiveness of treatment for IL-12 related disorders. (Kiniwa etal. J. Clin. Invest., 90: 262-66, 1992)

6.7.13. Leukemias

Leukemias and related disorders may be treated or prevented byadministration of a therapeutic that promotes or inhibits function ofthe polynucleotides and/or polypeptides of the invention. Such leukemiasand related disorders include but are not limited to acute leukemia,acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic,promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronicleukemia, chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia (for a review of such disorders, see Fishman etal., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia).

6.7.14. Nervous System Disorders

Nervous system disorders, involving cell types which can be tested forefficacy of intervention with compounds that modulate the activity ofthe polynucleotides and/or polypeptides of the invention, and which canbe treated upon thus observing an indication of therapeutic utility,include but are not limited to nervous system injuries, and diseases ordisorders which result in either a disconnection of axons, a diminutionor degeneration of neurons, or demyelination. Nervous system lesionswhich may be treated in a patient (including human and non-humanmammalian patients) according to the invention include but are notlimited to the following lesions of either the central (including spinalcord, brain) or peripheral nervous systems:

(i) traumatic lesions, including lesions caused by physical injury orassociated with surgery, for example, lesions which sever a portion ofthe nervous system, or compression injuries;

(ii) ischemic lesions, in which a lack of oxygen in a portion of thenervous system results in neuronal injury or death, including cerebralinfarction or ischemia, or spinal cord infarction or ischemia;

(iii) infectious lesions, in which a portion of the nervous system isdestroyed or injured as a result of infection, for example, by anabscess or associated with infection by human immunodeficiency virus,herpes zoster, or herpes simplex virus or with Lyme disease,tuberculosis, syphilis;

(iv) degenerative lesions, in which a portion of the nervous system isdestroyed or injured as a result of a degenerative process including butnot limited to degeneration associated with Parkinson's disease,Alzheimer's disease, Huntington's chorea, or amyotrophic lateralsclerosis;

(v) lesions associated with nutritional diseases or disorders, in whicha portion of the nervous system is destroyed or injured by a nutritionaldisorder or disorder of metabolism including but not limited to, vitaminB12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcoholamblyopia, Marchiafava-Bignami disease (primary degeneration of thecorpus callosum), and alcoholic cerebellar degeneration;

(vi) neurological lesions associated with systemic diseases includingbut not limited to diabetes (diabetic neuropathy, Bell's palsy),systemic lupus erythematosus, carcinoma, or sarcoidosis;

(vii) lesions caused by toxic substances including alcohol, lead, orparticular neurotoxins; and

(viii) demyelinated lesions in which a portion of the nervous system isdestroyed or injured by a demyelinating disease including but notlimited to multiple sclerosis, human immunodeficiency virus-associatedmyelopathy, transverse myelopathy or various etiologies, progressivemultifocal leukoencephalopathy, and central pontine myelinolysis.

Therapeutics which are useful according to the invention for treatmentof a nervous system disorder may be selected by testing for biologicalactivity in promoting the survival or differentiation of neurons. Forexample, and not by way of limitation, therapeutics which elicit any ofthe following effects may be useful according to the invention:

(i) increased survival time of neurons in culture;

(ii) increased sprouting of neurons in culture or in vivo;

(iii) increased production of a neuron-associated molecule in culture orin vivo, e.g., choline acetyltransferase or acetylcholinesterase withrespect to motor neurons; or

(iv) decreased symptoms of neuron dysfunction in vivo. Such effects maybe measured by any method known in the art. In preferred, non-limitingembodiments, increased survival of neurons may be measured by the methodset forth in Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increasedsprouting of neurons may be detected by methods set forth in Pestronk etal. (1980, Exp. Neurol. 70:65-82) or Brown et al. (1981, Ann. Rev.Neurosci. 4:17-42); increased production of neuron-associated moleculesmay be measured by bioassay, enzymatic assay, antibody binding, Northernblot assay, etc., depending on the molecule to be measured; and motorneuron dysfunction may be measured by assessing the physicalmanifestation of motor neuron disorder, e.g., weakness, motor neuronconduction velocity, or functional disability.

In a specific embodiments, motor neuron disorders that may be treatedaccording to the invention include but are not limited to disorders suchas infarction, infection, exposure to toxin, trauma, surgical damage,degenerative disease or malignancy that may affect motor neurons as wellas other components of the nervous system, as well as disorders thatselectively affect neurons such as amyotrophic lateral sclerosis, andincluding but not limited to progressive spinal muscular atrophy,progressive bulbar palsy, primary lateral sclerosis, infantile andjuvenile muscular atrophy, progressive bulbar paralysis of childhood(Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, andHereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

6.7.15. Other Activities

A protein of the invention may also exhibit one or more of the followingadditional activities or effects: inhibiting the growth, infection orfunction of, or killing, infectious agents, including, withoutlimitation, bacteria, viruses, fungi and other parasites; effecting(suppressing or enhancing) bodily characteristics, including, withoutlimitation, height, weight, hair color, eye color, skin, fat to leanratio or other tissue pigmentation, or organ or body part size or shape(such as, for example, breast augmentation or diminution, change in boneform or shape); effecting biorhythms or caricadic cycles or rhythms;effecting the fertility of male or female subjects; effecting themetabolism, catabolism, anabolism, processing, utilization, storage orelimination of dietary fat, lipid, protein, carbohydrate, vitamins,minerals, co-factors or other nutritional factors or component(s);effecting behavioral characteristics, including, without limitation,appetite, libido, stress, cognition (including cognitive disorders),depression (including depressive disorders) and violent behaviors;providing analgesic effects or other pain reducing effects; promotingdifferentiation and growth of embryonic stem cells in lineages otherthan hematopoietic lineages; hormonal or endocrine activity; in the caseof enzymes, correcting deficiencies of the enzyme and treatingdeficiency-related diseases; treatment of hyperproliferative disorders(such as, for example, psoriasis); immunoglobulin-like activity (suchas, for example, the ability to bind antigens or complement); and theability to act as an antigen in a vaccine composition to raise an immuneresponse against such protein or another material or entity which iscross-reactive with such protein.

6.8. Therapeutic Methods

The novel Interleukin-1 Receptor Antagonist of the invention hasnumerous applications in a variety of therapeutic methods. Examples oftherapeutic applications include, but are not limited to, thoseexemplified below.

6.8.1 Sepsis

One embodiment of the invention is the administration of an effectiveamount of the Interleukin-1 receptor antagonist polypeptides of theinvention to individuals that are at a high risk of developing sepsis,or that have developed sepsis. An example of the former category arepatients about to undergo surgery. While the mode of administration isnot particularly important, parenteral administration is preferredbecause of the rapid progression of sepsis, and thus, the need to havethe inhibitor disseminate quickly throughout the body. Thus, thepreferred mode of administration is to deliver an I.V. bolus slightlybefore, during, or after surgery. The dosage of the Interleukin-1receptor antagonist polypeptides of the invention will normally bedetermined by the prescribing physician. It is to be expected that thedosage will vary according to the age, weight and response of theindividual patient. Typically, the amount of inhibitor administered perdose will be in the range of about 0.1 to 25 mg/kg of body weight, withthe preferred dose being about 0.1 to 10 mg/kg of patient body weight.For parenteral administration, the Interleukin-1 receptor antagonistpolypeptides of the invention will be formulated in an injectable formcombined with a pharmaceutically acceptable parenteral vehicle. Suchvehicles are well known in the art and examples include water, saline,Ringer's solution, dextrose solution, and solutions consisting of smallamounts of the human serum albumin. The vehicle may contain minoramounts of additives that maintain the isotonicity and stability of theinhibitor. The preparation of such solutions is within the skill of theart. Typically, the cytokine inhibitor will be formulated in suchvehicles at a concentration of about 1-8 mg/ml to about 10 mg/ml.

6.8.2 Arthritis and Inflammation

The immunosuppressive effects of the Interleukin-1 inhibitor againstrheumatoid arthritis is determined in an experimental animal modelsystem. The experimental model system is adjuvant induced arthritis inrats, and the protocol is described by J. Holoshitz, et at., 1983,Science, 219:56, or by B. Waksman et al., 1963, Int. Arch. Allergy Appl.Immunol., 23:129. Induction of the disease can be caused by a singleinjection, generally intradermally, of a suspension of killedMycobacterium tuberculosis in complete Freund's adjuvant (CFA). Theroute of injection can vary, but rats may be injected at the base of thetail with an adjuvant mixture. The inhibitor is administered inphosphate buffered solution (PBS) at a dose of about 1-5 mg/kg. Thecontrol consists of administering PBS only.

The procedure for testing the effects of the Interleukin-1 inhibitorwould consist of intradermally injecting killed Mycobacteriumtuberculosis in CFA followed by immediately administering the inhibitorand subsequent treatment every other day until day 24. At 14, 15, 18,20, 22, and 24 days after injection of Mycobacterium CFA, an overallarthritis score may be obtained as described by J. Holoskitz above. Ananalysis of the data would reveal that the inhibitor would have adramatic affect on the swelling of the joints as measured by a decreaseof the arthritis score.

6.8.3 Diabetes

Interleukin-1 has been shown to be involved in the destruction of isletcells in diabetes mellitus (DM) (Mandrup-Paulsen, T., K. Bendtzen, J.Nerup, C. A. Dinarello, M. Svenson, and J. H. Nielson [1986]Diabetologia 29:63-67). The Interleukin-1 receptor antagonistpolypeptides of the invention limit lymphocyte and macrophage mediateddamage to islet cells in incipient cases of DM identified by diseasesusceptibility via genetic background and family history. Theinflammatory destruction of the pancreatic beta islet cells in suchindividuals with early DM is reduced by parenterally administering theInterleukin-1 receptor antagonist polypeptides of the invention whichhave an anti-Interleukin-1 effect in the pancreas.

6.8.4 Anti-Hypotensive Arginine-Free Formulations

The parenteral formulation of the therapeutic regimen is defined asincluding: about 3-4 g/l isoleucine, about 4-6 g/l leucine, about 3-4g/l lysine, about 1-2 g/l methionine, about 1-2 g/l phenylalanine, about2-3 g/l threonine, about 0.5-1.5 g/l tryptophan, about 3-4 g/l valine,about 4-5 g/l alanine, about 1-2 g/l histidine, about 3-4 g/l proline,about 1-2 g/l serine, about 0.25-0.75 g/l tyrosine, about 4-5 g/lglycine and about 2-3 g/l aspartic acid, together in a pharmacologicallyacceptable excipient. In another preferred embodiment of the describedparenteral formulation, the formulation may further include ornithine,most particularly at a concentration of about 1-2 g/l. In still anotherembodiment of the described parenteral formulation, the formulation mayinclude citrulline, most preferably at a concentration of between about1 g/l and about 2 g/l. Both citrulline and ornithine may be included instill another embodiment of the formulation, again at the concentrationsindicated.

The method includes an arginine-free formulation which comprises theamino acids and concentrations thereof already described herein,together in a pharmacologically acceptable excipient. Again, theformulation may further include ornithine, citrulline, or both, to evenfurther supply physiologically required concentrations of urea cyclesubstrates in the animal. Most preferably, the formulation is providedas a parenteral formulation.

Another aspect of the method comprises a method for treatingchemotherapeutic agent-related hypotension. In a most preferredembodiment, the method comprises monitoring an animal receiving achemotherapeutic agent for a decrease in systolic blood pressure to lessthan about 100 mm Hg to detect an animal with systemic hypotension,treating the animal having systemic hypotension with a therapeuticregimen comprising a therapeutically effective amount of anarginine-free formulation sufficient to reduce plasma or serum arginineconcentrations administered concurrently with or followed by theadministration of a therapeutically effective concentration of aninterleukin-1 receptor antagonist, and maintaining the animal on thetherapeutic regimen until an increase of systolic blood pressure to atleast about 100 mm Hg is detectable. Most preferably, the arginine-freeformulation is a parenteral formulation.

In a preferred embodiment, the interleukin-1 receptor antagonistpolypeptide of the invention is used in combination with theanti-hypotensive arginine free formulation to treat hypotension in ananimal, particularly that hypotension caused by exposure to endotoxin orseptic shock.

A patient having a systolic blood pressure of less than about 100 mm Hgwill be targeted for the present treatment. Such a patient is to beplaced on a continuous feed of an arginine-free formulation whichincludes a mixture of essential and nonessential amino acids asdescribed in U.S. Pat. No. 5,334,380. The patient is treatedconcurrently with the interleukin-1 Hy1 receptor antagonist polypeptidesof the invention. Blood samples are to be obtained from the patient andarginine levels in the serum or plasma fraction are determined.

6.9 Pharmaceutical Formulations and Routes of Administration

A protein of the present invention (from whatever source derived,including without limitation from recombinant and non-recombinantsources) may be administered to a patient in need, by itself, or inpharmaceutical compositions where it is mixed with suitable carriers orexcipient(s) at doses to treat or ameliorate a variety of disorders.Such a composition may also contain (in addition to protein and acarrier) diluents, fillers, salts, buffers, stabilizers, solubilizers,and other materials well known in the art. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).The characteristics of the carrier will depend on the route ofadministration. The pharmaceutical composition of the invention may alsocontain cytokines, lymphokines, or other hematopoietic factors such asM-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, G-CSF, Meg-CSF,thrombopoietin, stem cell factor, and erythropoietin.

The pharmaceutical composition may further contain other agents whicheither enhance the activity of the protein or complement its activity oruse in treatment. Such additional factors and/or agents may be includedin the pharmaceutical composition to produce a synergistic effect withprotein of the invention, or to minimize side effects. Proteins that canbe administered with IL-1Hy1 include other IL-1 receptor antagonistpolypeptides such as IL-1ra and IL-1Hy2, described in U.S. Ser. No.09/316,081 filed May 20, 1999. Conversely, protein of the presentinvention may be included in formulations of the particular cytokine,lymphokine, other hematopoietic factor, thrombolytic or anti-thromboticfactor, or anti-inflammatory agent to minimize side effects of thecytokine, lymphokine, other hematopoietic factor, thrombolytic oranti-thrombotic factor, or anti-inflammatory agent. A protein of thepresent invention may be active in multimers (e.g., heterodimers orhomodimers) or complexes with itself or other proteins. As a result,pharmaceutical compositions of the invention may comprise a protein ofthe invention in such multimeric or complexed form.

Techniques for formulation and administration of the compounds of theinstant application may be found in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., latest edition. Atherapeutically effective dose further refers to that amount of thecompound sufficient to result in amelioration of symptoms, e.g.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, a therapeutically effective dose refersto that ingredient alone. When applied to a combination, atherapeutically effective dose refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredin combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of protein of the present invention isadministered to a mammal having a condition to be treated. Protein ofthe present invention maybe administered in accordance with the methodof the invention either alone or in combination with other therapiessuch as treatments employing cytokines, lymphokines or otherhematopoietic factors. When co-administered with one or more cytokines,lymphokines or other hematopoietic factors, protein of the presentinvention may be administered either simultaneously with thecytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolyticor anti-thrombotic factors, or sequentially. If administeredsequentially, the attending physician will decide on the appropriatesequence of administering protein of the present invention incombination with cytokine(s), lymphokine(s), other hematopoietic.factor(s), thrombolytic or anti-thrombotic factors.

6.9.1. Routes of Administration

Suitable routes of administration may, for example, include oral,rectal, transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections. Administrationof protein of the present invention used in the pharmaceuticalcomposition or to practice the method of the present invention can becarried out in a variety of conventional ways, such as oral ingestion,inhalation, topical application or cutaneous, subcutaneous,intraperitoneal, parenteral or intravenous injection. Intravenousadministration to the patient is preferred.

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a arthritic joints or in fibrotic tissue, often in a depot orsustained release formulation. In order to prevent the scarring processfrequently occurring as complication of glaucoma surgery, the compoundsmay be administered topically, for example, as eye drops. Furthermore,one may administer the drug in a targeted drug delivery system, forexample, in a liposome coated with a specific antibody, targeting, forexample, arthritic or fibrotic tissue. The liposomes will be targeted toand taken up selectively by the afflicted tissue.

6.9.2. Compositions/Formulations

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in a conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. These pharmaceuticalcompositions may be manufactured in a manner that is itself known, e.g.,by means of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Proper formulation is dependent upon the route ofadministration chosen. When a therapeutically effective amount ofprotein of the present invention is administered orally, protein of thepresent invention will be in the form of a tablet, capsule, powder,solution or elixir. When administered in tablet form, the pharmaceuticalcomposition of the invention may additionally contain a solid carriersuch as a gelatin or an adjuvant. The tablet, capsule, and powdercontain from about 5 to 95% protein of the present invention, andpreferably from about 25 to 90% protein of the present invention. Whenadministered in liquid form, a liquid carrier such as water, petroleum,oils of animal or plant origin such as peanut. oil, mineral oil, soybeanoil, or sesame oil, or synthetic oils may be added. The liquid form ofthe pharmaceutical composition may further contain physiological salinesolution, dextrose or other saccharide solution, or glycols such asethylene glycol, propylene glycol or polyethylene glycol. Whenadministered in liquid form, the pharmaceutical composition containsfrom about 0.5 to 90% by weight of protein of the present invention, andpreferably from about 1 to 50% protein of the present invention.

When a therapeutically effective amount of protein of the presentinvention is administered by intravenous, cutaneous or subcutaneousinjection, protein of the present invention will be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such parenterally acceptable protein solutions, having due regard topH, isotonicity, stability, and the like, is within the skill in theart. A preferred pharmaceutical composition for intravenous, cutaneous,or subcutaneous injection should contain, in addition to protein of thepresent invention, an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art. Forinjection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration. For buccal administration, the compositions may take theform of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch. The compounds maybe formulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides. In additionto the formulations described previously, the compounds may also beformulated as a depot preparation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

A pharmaceutical carrier for the hydrophobic compounds of the inventionis a cosolvent system comprising benzyl alcohol, a nonpolar surfactant,a water-miscible organic polymer, and an aqueous phase. The cosolventsystem may be the VPD co-solvent system. VPD is a solution of 3% w/vbenzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5%dextrose in water solution. This co-solvent system dissolves hydrophobiccompounds well, and itself produces low toxicity upon systemicadministration. Naturally, the proportions of a co-solvent system may bevaried considerably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied: for example, other low-toxicity nonpolar surfactants maybe used instead of polysorbate 80; the fraction size of polyethyleneglycol may be varied; other biocompatible polymers may replacepolyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars orpolysaccharides may substitute for dextrose. Alternatively, otherdelivery systems for hydrophobic pharmaceutical compounds may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs. Certain organic solventssuch as dimethylsulfoxide also may be employed, although usually at thecost of greater toxicity. Additionally, the compounds may be deliveredusing a sustained-release system, such as semipermeable matrices ofsolid hydrophobic polymers containing the therapeutic agent. Various ofsustained-release materials have been established and are well known bythose skilled in the art. Sustained-release capsules may, depending ontheir chemical nature, release the compounds for a few weeks up to over100 days. Depending on the chemical nature and the biological stabilityof the therapeutic reagent, additional strategies for proteinstabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols. Many of the compounds of the invention maybe provided as salts with pharmaceutically compatible counterions. Suchpharmaceutically acceptable base addition salts are those salts whichretain the biological effectiveness and properties of the free acids andwhich are obtained by reaction with inorganic or organic bases such assodium hydroxide, magnesium hydroxide, ammonia, trialkylamine,dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate,potassium benzoate, triethanol amine and the like.

The pharmaceutical composition of the invention may be in the form of acomplex of the protein(s) of present invention along with protein orpeptide antigens. The protein and/or peptide antigen will deliver astimulatory signal to both B and T lymphocytes. B lymphocytes willrespond to antigen through their surface immunoglobulin receptor. Tlymphocytes will respond to antigen through the T cell receptor (TCR)following presentation of the antigen by MHC proteins. MHC andstructurally related proteins including those encoded by class I andclass II MHC genes on host cells will serve to present the peptideantigen(s) to T lymphocytes. The antigen components could also besupplied as purified MHC-peptide complexes alone or with co-stimulatorymolecules that can directly signal T cells. Alternatively antibodiesable to bind surface immunoglobulin and other molecules on B cells aswell as antibodies able to bind the TCR and other molecules on T cellscan be combined with the pharmaceutical composition of the invention.The pharmaceutical composition of the invention may be in the form of aliposome in which protein of the present invention is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids which exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers in aqueoussolution. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. Preparation of suchliposomal formulations is within the level of skill in the art, asdisclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728;4,837,028; and 4,737,323, all of which are incorporated herein byreference.

The amount of protein of the present invention in the pharmaceuticalcomposition of the present invention will depend upon the nature andseverity of the condition being treated, and on the nature of priortreatments which the patient has undergone. Ultimately, the attendingphysician will decide the amount of protein of the present inventionwith which to treat each individual patient. Initially, the attendingphysician will administer low doses of protein of the present inventionand observe the patient's response. Larger doses of protein of thepresent invention may be administered until the optimal therapeuticeffect is obtained for the patient, and at that point the dosage is notincreased further. It is contemplated that the various pharmaceuticalcompositions used to practice the method of the present invention shouldcontain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about10 mg, more preferably about 0.1 μg to about 1 mg) of protein of thepresent invention per kg body weight. For compositions of the presentinvention which are useful for bone, cartilage, tendon or ligamentregeneration, the therapeutic method includes administering thecomposition topically, systematically, or locally as an implant ordevice. When administered, the therapeutic composition for use in thisinvention is, of course, in a pyrogen-free, physiologically acceptableform. Further, the composition may desirably be encapsulated or injectedin a viscous form for delivery to the site of bone, cartilage or tissuedamage. Topical administration may be suitable for wound healing andtissue repair. Therapeutically useful agents other than a protein of theinvention which may also optionally be included in the composition asdescribed above, may alternatively or additionally, be administeredsimultaneously or sequentially with the composition in the methods ofthe invention. Preferably for bone and/or cartilage formation, thecomposition would include a matrix capable of delivering theprotein-containing composition to the site of bone and/or cartilagedamage, providing a structure for the developing bone and cartilage andoptimally capable of being resorbed into the body. Such matrices may beformed of materials presently in use for other implanted medicalapplications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid,polyglycolic acid and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sinteredhydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may becomprised of combinations of any of the above mentioned types ofmaterial, such as polylactic acid and hydroxyapatite or collagen andtricalciumphosphate. The bioceramics may be altered in composition, suchas in calcium-aluminate-phosphate and processing to alter pore size,particle size, particle shape, and biodegradability. Presently preferredis a 50:50 (mole weight) copolymer of lactic acid and glycolic acid inthe form of porous particles having diameters ranging from 150 to 800microns. In some applications, it will be useful to utilize asequestering agent, such as carboxymethyl cellulose or autologous bloodclot, to prevent the protein compositions from disassociating from thematrix.

A preferred family of sequestering agents is cellulosic materials suchas alkylcelluloses (including hydroxyalkylcelluloses), includingmethylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropyl-methylcellul se, andcarboxymethylcellulose, the most preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate, poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). Theamount of sequestering agent useful herein is 0.5-20 wt %, preferably1-10 wt % based on total formulation weight, which represents the amountnecessary to prevent desorbtion of the protein from the polymer matrixand to provide appropriate handling of the composition, yet not so muchthat the progenitor cells are prevented from infiltrating the matrix,thereby providing the protein the opportunity to assist the osteogenicactivity of the progenitor cells. In further compositions, proteins ofthe invention may be combined with other agents beneficial to thetreatment of the bone and/or cartilage defect, wound, or tissue inquestion. These agents include various growth factors such as epidermalgrowth factor (EGF), platelet derived growth factor (PDGF), transforminggrowth factors (TGF-.alpha. and TGF-.beta.), and insulin-like growthfactor (IGF).

The therapeutic compositions are also presently valuable for veterinaryapplications. Particularly domestic animals and thoroughbred horses, inaddition to humans, are desired patients for such treatment withproteins of the present invention. The dosage regimen of aprotein-containing pharmaceutical composition to be used in tissueregeneration will be determined by the attending physician consideringvarious factors which modify the action of the proteins, e.g., amount oftissue weight desired to be formed, the site of damage, the condition ofthe damaged tissue, the size of a wound, type of damaged tissue (e.g.,bone), the patient's age, sex, and diet, the severity of any infection,time of administration and other clinical factors. The dosage may varywith the type of matrix used in the reconstitution and with inclusion ofother proteins in the pharmaceutical composition. For example, theaddition of other known growth factors, such as IGF I (insulin likegrowth factor I), to the final composition, may also effect the dosage.Progress can be monitored by periodic assessment of tissue/bone growthand/or repair, for example, X-rays, histomorphometric determinations andtetracycline labeling.

Polynucleotides of the present invention can also be used for genetherapy. Such polynucleotides can be introduced either in vivo or exvivo into cells for expression in a mammalian subject. Polynucleotidesof the invention may also be administered by other known methods forintroduction of nucleic acid into a cell or organism (including, withoutlimitation, in the form of viral vectors or naked DNA). Cells may alsobe cultured ex vivo in the presence of proteins of the present inventionin order to proliferate or to produce a desired effect on or activity insuch cells. Treated cells can then be introduced in vivo for therapeuticpurposes.

6.9.3. Effective Dosage

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. More specifically, atherapeutically effective amount means an amount effective to preventdevelopment of or to alleviate the existing symptoms of the subjectbeing treated. Determination of the effective amounts is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein. For any compound used in the methodof the invention, the therapeutically effective dose can be estimatedinitially from cell culture assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms or a prolongation of survivalin a patient. Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratiobetween LD₅₀ and ED₅₀. Compounds which exhibit high therapeutic indicesare preferred. The data obtained from these cell culture assays andanimal studies can be used in formulating a range of dosage for use inhuman. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. See, e.g.,Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1 p. 1. Dosage amount and interval may be adjusted individually toprovide plasma levels of the active moiety which are sufficient tomaintain the desired therapeutic effects, or minimal effectiveconcentration (MEC). The MEC will vary for each compound but can beestimated from in vitro data; for example, the concentration necessaryto achieve 50-90% inhibition of cytokine activity using the assaysdescribed herein. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. However, HPLCassays or bioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen which maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%. In cases of local administration or selectiveuptake, the effective local concentration of the drug may not be relatedto plasma concentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

6.9.4. Packaging

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may, for example, comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. Compositions comprisinga compound of the invention formulated in a compatible pharmaceuticalcarrier may also be prepared, placed in an appropriate container, andlabeled for treatment of an indicated condition.

6.10. Antibodies

Another aspect of the invention is an antibody that specifically bindsthe polypeptide of the invention. Such antibodies can be eithermonoclonal or polyclonal antibodies, as well fragments thereof andhumanized forms or fully human forms, such as those produced intransgenic animals. The invention further provides a hybridoma thatproduces an antibody according to the invention. Antibodies of theinvention are useful for detection and/or purification of thepolypeptides of the invention.

Protein of the invention may also be used to immunize animals to obtainpolyclonal and monoclonal antibodies which specifically react with theprotein. Such antibodies may be obtained using either the entire proteinor fragments thereof as an immunogen. The peptide immunogensadditionally may contain a cysteine residue at the carboxyl terminus,and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH).Methods for synthesizing such peptides are known in the art, forexample, as in R. P. Merrifield, J. Amer. Chem. Soc. 85, 2149-2154(1963); J. L. Krstenansky, et al., FEBS Lett. 211, 10 (1987). Monoclonalantibodies binding to the protein of the invention may be usefuldiagnostic agents for the immunodetection of the protein. Neutralizingmonoclonal antibodies binding to the protein may also be usefultherapeutics for both conditions associated with the protein and also inthe treatment of some forms of cancer where abnormal expression of theprotein is involved. In the case of cancerous cells or leukemic cells,neutralizing monoclonal antibodies against the protein may be useful indetecting and preventing the metastatic spread of the cancerous cells,which may be mediated by the protein. In general, techniques forpreparing polyclonal and monoclonal antibodies as well as hybridomascapable of producing the desired antibody are well known in the art(Campbell, A. M., Monoclonal Antibodies Technology: LaboratoryTechniques in Biochemistry and Molecular Biology, Elsevier SciencePublishers, Amsterdam, The Netherlands (1984); St. Groth et al., J.Immunol. 35:1-21 (1990); Kohler and Milstein, Nature 256:495-497(1975)), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., Immunology Today 4:72 (1983); Cole et al., in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), pp. 77-96).

Any animal (mouse, rabbit, etc.) which is known to produce antibodiescan be immunized with a peptide or polypeptide of the invention. Methodsfor immunization are well known in the art. Such methods includesubcutaneous or intraperitoneal injection of the polypeptide. Oneskilled in the art will recognize that the amount of the protein encodedby the ORF of the present invention used for immunization will varybased on the animal which is immunized, the antigenicity of the peptideand the site of injection. The protein that is used as an immunogen maybe modified or administered in an adjuvant in order to increase theprotein's antigenicity. Methods of increasing the antigenicity of aprotein are well known in the art and include, but are not limited to,coupling the antigen with a heterologous protein (such as globulin orβ-galactosidase) or through the inclusion of an adjuvant duringimmunization.

For monoclonal antibodies, spleen cells from the immunized animals areremoved, fused with myeloma cells, such as SP2/0-Ag14 myeloma cells, andallowed to become monoclonal antibody producing hybridoma cells. Any oneof a number of methods well known in the art can be used to identify thehybridoma cell which produces an antibody with the desiredcharacteristics. These include screening the hybridomas with an ELISAassay, western blot analysis, or radioimmunoassay (Lutz et al., Exp.Cell Research. 175:109-124 (1988)).

Hybridomas secreting the desired antibodies are cloned and the class andsubclass is determined using procedures known in the art (Campbell, A.M., Monoclonal Antibody Technology: Laboratory Techniques inBiochemistry and Molecular Biology, Elsevier Science Publishers,Amsterdam, The Netherlands (1984)). Techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778) can beadapted to produce single chain antibodies to proteins of the presentinvention.

For polyclonal antibodies, antibody containing antiserum is isolatedfrom the immunized animal and is screened for the presence of antibodieswith the desired specificity using one of the above-describedprocedures. The present invention further provides the above-describedantibodies in delectably labeled form. Antibodies can be delectablylabeled through the use of radioisotopes, affinity labels (such asbiotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase,alkaline phosphatase, etc.) fluorescent labels (such as FITC orrhodamine, etc.), paramagnetic atoms, etc. Procedures for accomplishingsuch labeling are well-known in the art, for example, see (Stemberger,L. A. et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E. A. etal., Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129(1972); Goding, J. W. J. Immunol. Meth. 13:215 (1976)).

The labeled antibodies of the present invention can be used for invitro, in vivo, and in situ assays to identify cells or tissues in whicha fragment of the polypeptide of interest is expressed. The antibodiesmay also be used directly in therapies or other diagnostics. The presentinvention further provides the above-described antibodies immobilized ona solid support. Examples of such solid supports include plastics suchas polycarbonate, complex carbohydrates such as agarose and sepharose,acrylic resins and such as polyacrylamide and latex beads. Techniquesfor coupling antibodies to such solid supports are well known in the art(Weir, D. M. et al., “Handbook of Experimental Immunology” 4th Ed.,Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986);Jacoby, W. D. et al., Meth. Enzym. 34 Academic Press, N.Y. (1974)). Theimmobilized antibodies of the present invention can be used for invitro, in vivo, and in situ assays as well as for immuno-affinitypurification of the proteins of the present invention.

6.11. Computer Readable Sequences

In one application of this embodiment, a nucleotide sequence of thepresent invention can be recorded on computer readable media. As usedherein, “computer readable media” refers to any medium which can be readand accessed directly by a computer. Such media include, but are notlimited to: magnetic storage media, such as floppy discs, hard discstorage medium, and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; and hybrids of thesecategories such as magnetic/optical storage media. A skilled artisan canreadily appreciate how any of the presently known computer readablemediums can be used to create a manufacture comprising computer readablemedium having recorded thereon a nucleotide sequence of the presentinvention. As used herein, “recorded” refers to a process for storinginformation on computer readable medium. A skilled artisan can readilyadopt any of the presently known methods for recording information oncomputer readable medium to generate manufactures comprising thenucleotide sequence information of the present invention.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon anucleotide sequence of the present invention. The choice of the datastorage structure will generally be based on the means chosen to accessthe stored information. In addition, a variety of data processorprograms and formats can be used to store the nucleotide sequenceinformation of the present invention on computer readable medium. Thesequence information can be represented in a word processing text file,formatted in commercially-available software such as WordPerfect andMicrosoft Word, or represented in the form of an ASCII file, stored in adatabase application, such as DB2, Sybase, Oracle, or the like. Askilled artisan can readily adapt any number of dataprocessorstructuring formats (e.g. text file or database) in order to obtaincomputer readable medium having recorded thereon the nucleotide sequenceinformation of the present invention. By providing the nucleotidesequence of SEQ ID NOS: 1, 2, 4, 6 or 7 or a representative fragmentthereof, or a nucleotide sequence at least 99.9% identical to SEQ IDNOS: 1, 2 , 4, 5, 6 or 7 or 8 in computer readable form, a skilledartisan can routinely access the sequence information for a variety ofpurposes. Computer software is publicly available which allows a skilledartisan to access sequence information provided in a computer readablemedium. The examples which follow demonstrate how software whichimplements the BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990))and BLAZE (Brutlag et al., Comp. Chem. 17:203-207 (1993)) searchalgorithms on a Sybase system is used to identify open reading frames(ORFs) within a nucleic acid sequence. Such ORFs may be protein encodingfragments and may be useful in producing commercially important proteinssuch as enzymes used in fermentation reactions and in the production ofcommercially useful metabolites.

As used herein, “a computer-based system” refers to the hardware means,software means, and data storage means used to analyze the nucleotidesequence information of the present invention. The minimum hardwaremeans of the computer-based systems of the present invention comprises acentral processing unit (CPU), input means, output means, and datastorage means. A skilled artisan can readily appreciate that any one ofthe currently available computer-based systems are suitable for use inthe present invention. As stated above, the computer-based systems ofthe present invention comprise a data storage means having storedtherein a nucleotide sequence of the present invention and the necessaryhardware means and software means for supporting and implementing asearch means. As used herein, “data storage means” refers to memorywhich can store nucleotide sequence information of the presentinvention, or a memory access means which can access manufactures havingrecorded thereon the nucleotide sequence information of the presentinvention.

As used herein, “search means” refers to one or more programs which areimplemented on the computer-based system to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. Search means are used to identify fragments orregions of a known sequence which match a particular target sequence ortarget motif. A variety of known algorithms are disclosed publicly and avariety of commercially available software for conducting search meansare and can be used in the computer-based systems of the presentinvention. Examples of such software includes, but is not limited to,MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). A skilled artisancan readily recognize that any one of the available algorithms orimplementing software packages for conducting homology searches can beadapted for use in the present computer-based systems. As used herein, a“target sequence” can be any nucleic acid or amino acid sequence of sixor more nucleotides or two or more amino acids. A skilled artisan canreadily recognize that the longer a target sequence is, the less likelya target sequence will be present as a random occurrence in thedatabase. The most preferred sequence length of a target sequence isfrom about 10 to 100 amino acids or from about 30 to 300 nucleotideresidues. However, it is well recognized that searches for commerciallyimportant fragments, such as sequence fragments involved in geneexpression and protein processing, may be of shorter length.

As used herein, “a target structural motif,” or “target motif,” refersto any rationally selected sequence or combination of sequences in whichthe sequence(s) are chosen based on a three-dimensional configurationwhich is formed upon the folding of the target motif. There are avariety of target motifs known in the art. Protein target motifsinclude, but are not limited to, enzyme active sites and signalsequences. Nucleic acid target motifs include, but are not limited to,promoter sequences, hairpin structures and inducible expression elements(protein binding sequences).

6.12. Triple Helix Formation

In addition, the fragments of the present invention, as broadlydescribed, can be used to control gene expression through triple helixformation or antisense DNA or RNA, both of which methods are based onthe binding of a polynucleotide sequence to DNA or RNA. Polynucleotidessuitable for use in these methods are usually 20 to 40 bases in lengthand are designed to be complementary to a region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 15241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Olmno, J.Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triplehelix—formation optimally results in a shut-off of RNA transcriptionfrom DNA, while antisense RNA hybridization blocks translation of anmRNA molecule into polypeptide. Both techniques have been demonstratedto be effective in model systems. Information contained in the sequencesof the present invention is necessary for the design of an antisense ortriple helix oligonucleotide.

6.13. Diagnostic Assay and Kits

The present invention further provides methods to identify the presenceor expression of one of the ORFs of the present invention, or homologthereof, in a test sample, using a nucleic acid probe or antibodies ofthe present invention.

In general, methods for detecting a polynucleotide of the invention cancomprise contacting a sample with a compound that binds to and forms acomplex with the polynucleotide for a period sufficient to form thecomplex, and detecting the complex, so that if a complex is detected, apolynucleotide of the invention is detected in the sample. Such methodscan also comprise contacting a sample under stringent hybridizationconditions with nucleic acid primers that anneal to a polynucleotide ofthe invention under such conditions, and amplifying annealedpolynucleotides, so that if a polynucleotide is amplified, apolynucleotide of the invention is detected in the sample.

In general, methods for detecting a polypeptide of the invention cancomprise contacting a sample with a compound that binds to and forms acomplex with the polypeptide for a period sufficient to form thecomplex, and detecting the complex, so that if a complex is detected, apolypeptide of the invention is detected in the sample. In detail, suchmethods comprise incubating a test sample with one or more of theantibodies or one or more of nucleic acid probes of the presentinvention and assaying for binding of the nucleic acid probes orantibodies to components within the test sample.

Conditions for incubating a nucleic acid probe or antibody with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid probe or antibody used in the assay. One skilled in the artwill recognize that any one of the commonly available hybridization,amplification or immunological assay formats can readily be adapted toemploy the nucleic acid probes or antibodies of the present invention.Examples of such assays can be found in Chard, T., An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985). The test samplesof the present invention include cells, protein or membrane extracts ofcells, or biological fluids such as sputum, blood, serum, plasma, orurine. The test sample used in the above-described method will varybased on the assay format, nature of the detection method and thetissues, cells or extracts used as the sample to be assayed. Methods forpreparing protein extracts or membrane extracts of cells are well knownin the art and can be readily be adapted in order to obtain a samplewhich is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention. Specifically, the invention provides a compartment kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the probes or antibodies of thepresent invention; and (b) one or more other containers comprising oneor more of the following: wash reagents, reagents capable of detectingpresence of a bound probe or antibody.

In detail, a compartment kit includes any kit in which reagents arecontained in separate containers. Such containers include small glasscontainers, plastic containers or strips of plastic or paper. Suchcontainers allows one to efficiently transfer reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated, and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother. Such containers will include a container which will accept thetest sample, a container which contains the antibodies used in theassay, containers which contain wash reagents (such as phosphatebuffered saline, Tris-buffers, etc.), and containers which contain thereagents used to detect the bound antibody or probe. Types of detectionreagents include labeled nucleic acid probes, labeled secondaryantibodies, or in the alternative, if the primary antibody is labeled,the enzymatic, or antibody binding reagents which are capable ofreacting with the labeled antibody. One skilled in the art will readilyrecognize that the disclosed probes and antibodies of the presentinvention can be readily incorporated into one of the established kitformats which are well known in the art.

6.14. Screening Assays

Using the isolated proteins and polynucleotides of the invention, thepresent invention further provides methods of obtaining and identifyingagents which bind to a polypeptide encoded by the. ORF from apolynucleotide with a sequence of SEQ ID NOS: 1, 2, 4, 6, 7 or 8 to aspecific domain of the polypeptide encoded by the nucleic acid, or to anucleic acid with a sequence of SEQ ID NOS: 1, 2 4, 6, 7 or 8. Indetail, said method comprises the steps of:

(a) contacting an agent with an isolated protein encoded by an ORF ofthe present invention, or nucleic acid of the invention; and

(b) determining whether the agent binds to said protein or said nucleicacid.

In general, therefore, such methods for identifying compounds that bindto a polynucleotide of the invention can comprise contacting a compoundwith a polynucleotide of the invention for a time sufficient to form apolynucleotide/compound complex, and detecting the complex, so that if apolynucleotide/compound complex is detected, a compound that binds to apolynucleotide of the invention is identified.

Likewise, in general, therefore, such methods for identifying compoundsthat bind to a polypeptide of the invention can comprise contacting acompound with a polypeptide of the invention for a time sufficient toform a polypeptide/compound complex, and detecting the complex, so thatif a polypeptide/compound complex is detected, a compound that binds toa polynucleotide of the invention is identified.

Methods for identifying compounds that bind to a polypeptide of theinvention can also comprise contacting a compound with a polypeptide ofthe invention in a cell for a time sufficient to form apolypeptide/compound complex, wherein the complex drives expression of areceptor gene sequence in the cell, and detecting the complex bydetecting reporter gene sequence expression, so that if apolypeptide/compound complex is detected, a compound that binds apolypeptide of the invention is identified.

Compounds identified via such methods can include compounds whichmodulate the activity of a polypeptide of the invention (that is,increase or decrease its activity, relative to activity observed in theabsence of the compound). Alternatively, compounds identified via suchmethods can include compounds which modulate the expression of apolynucleotide of the invention (that is, increase or decreaseexpression relative to expression levels observed in the absence of thecompound). Compounds, such as compounds identified via the methods ofthe invention, can be tested using standard assays well known to thoseof skill in the art for their ability to modulate activity/expression.

The agents screened in the above assay can be, but are not limited to,peptides, carbohydrates, vitamin derivatives, or other pharmaceuticalagents. The agents can be selected and screened at random or rationallyselected or designed using protein modeling techniques.

For random screening, agents such as peptides, carbohydrates,pharmaceutical agents and the like are selected at random and areassayed for their ability to bind to the protein encoded by the ORF ofthe present invention. Alternatively, agents may be rationally selectedor designed. As used herein, an agent is said to be “rationally selectedor designed” when the agent is chosen based on the configuration of theparticular protein. For example, one skilled in the art can readilyadapt currently. available procedures to generate peptides,pharmaceutical agents and the like capable of binding to a specificpeptide sequence in order to generate rationally designed antipeptidepeptides, for example see Hurby et al., Application of SyntheticPeptides: Antisense Peptides,” In Synthetic Peptides, A User's Guide, W.H. Freeman, NY (1992), pp. 289-307, and Kaspczak et al., Biochemistry28:9230-8 (1989), or pharmaceutical agents, or the like.

In addition to the foregoing, one class of agents of the presentinvention, as broadly described, can be used to control gene expressionthrough binding to one of the ORFs or EMFs of the present invention. Asdescribed above, such agents can be randomly screened or rationallydesigned/selected. Targeting the ORF or EMF allows a skilled artisan todesign sequence specific or element specific agents, modulating theexpression of either a single ORF or multiple ORFs which rely on thesame EMF for expression control. One class of DNA binding agents areagents which contain base residues which hybridize or form a triplehelix formation by binding to DNA or RNA. Such agents can be based onthe classic phosphodiester, ribonucleic acid backbone, or can be avariety of sulfhydryl or polymeric derivatives which have baseattachment capacity.

Agents suitable for use in these methods usually contain 20 to 40 basesand are designed to be complementary to a region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano. J.Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triplehelix—formation optimally results in a shut-off of RNA transcriptionfrom DNA, while antisense RNA hybridization blocks translation of anmRNA molecule into polypeptide. Both techniques have been demonstratedto be effective in model systems. Information contained in the sequencesof the present invention is necessary for the design of an antisense ortriple helix oligonucleotide and other DNA binding agents. Agents whichbind to a protein encoded by one of the ORFs of the present inventioncan be used as a diagnostic agent, in the control of bacterial infectionby modulating the activity of the protein encoded by the ORF. Agentswhich bind to a protein encoded by one of the ORFs of the presentinvention can be formulated using known techniques to generate apharmaceutical composition.

6.15. Use of Nucleic Acids as Probes

Another aspect of the subject invention is to provide forpolypeptide-specific nucleic acid hybridization probes capable ofhybridizing with naturally occurring nucleotide sequences. Thehybridization probes of the subject invention may be derived from thenucleotide sequence of the SEQ ID NOS: 1, 2, 4, 6, 7 or 8. Because thecorresponding gene is only expressed in a limited number of tissues,especially adult tissues, a hybridization probe derived from SEQ ID NOS:1, 2, 4, 6, 7, or 8 can be used as an indicator of the presence of RNAof cell type of such a tissue in a sample.

Any suitable hybridization technique can be employed, such as, forexample, in situ hybridization. PCR as described U.S. Pat. Nos 4,683,195and 4,965,188 provides additional uses for oligonucleotides based uponthe nucleotide sequences. Such probes used in PCR may be of recombinantorigin, may be chemically synthesized, or a mixture of both. The probewill comprise a discrete nucleotide sequence for the detection ofidentical sequences or a degenerate pool of possible sequences foridentification of closely related genomic sequences.

Other means for producing specific hybridization probes for nucleicacids include the cloning of nucleic acid sequences into vectors for theproduction of mRNA probes. Such vectors are known in the art and arecommercially available and may be used to synthesize RNA probes in vitroby means of the addition of the appropriate RNA polymerase as T7 or SP6RNA polymerase and the appropriate radioactively labeled nucleotides.The nucleotide sequences may be used to construct hybridization probesfor mapping their respective genomic sequences. The nucleotide sequenceprovided herein may be mapped to a chromosome or specific regions of achromosome using well known genetic and/or chromosomal mappingtechniques. These techniques include in situ hybridization, linkageanalysis against known chromosomal markers, hybridization screening withlibraries or flow-sorted chromosomal preparations specific to knownchromosomes, and the like. The technique of fluorescent in situhybridization of chromosome spreads has been described, among otherplaces, in Verma.et al (1988) Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York N.Y.

Fluorescent in situ hybridization of chromosomal preparations and otherphysical chromosome mapping techniques may be correlated with additionalgenetic map data. Examples of genetic map data can be found in the 1994Genome Issue of Science (265:1981f). Correlation between the location ofa nucleic acid on a physical chromosomal map and a specific disease (orpredisposition to a specific disease) may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier or affected individuals. The nucleotide sequencemay be used to produce purified polypeptides using well known methods ofrecombinant DNA technology. Among the many publications that teachmethods for the expression of genes after they have been isolated isGoeddel (1990) Gene Expression Technology, Methods and Enzymology, Vol185, Academic Press, San Diego. Polypeptides may be expressed in avariety of host cells, either prokaryotic or eukaryotic. Host cells maybe from the same species from which a particular polypeptide nucleotidesequence was isolated or from a different species. Advantages ofproducing polypeptides by recombinant DNA technology include obtainingadequate amounts of the protein for purification and the availability ofsimplified purification procedures.

14.1 Preparation of Sequencing Chips and Arrays

A basic example is using 6-mers attached to 50 micron surfaces to give achip with dimensions of 3×3 mm which can be combined to give an array of20×20 cm. Another example is using 9-mer oligonucleotides attached to10×10 microns surface to create a 9-mer chip, with dimensions of 5×5 mm.4000 units of such chips may be used to create a 30×30 cm array. In anarray in which 4,000 to 16,000 oligochips are arranged into a squarearray. A plate, or collection of tubes, as also depicted, may bepackaged with the array as part of the sequencing kit.

The arrays may be separated physically from each other or by hydrophobicsurfaces. One possible way to utilize the hydrophobic strip separationis to use technology such as the Iso-Grid Microbiology System producedby QA Laboratories, Toronto, Canada.

Hydrophobic grid membrane filters (HGMF) have been in use in analyticalfood microbiology for about a decade where they exhibit uniqueattractions of extended numerical range and automated counting ofcolonies. One commercially-available grid is ISO-GRID™ from QALaboratories Ltd. (Toronto, Canada) which consists of a square (60×60cm) of polysulfone polymer (Gelman Tuffryn HT-450, 0.45u pore size) onwhich is printed a black hydrophobic ink grid consisting of 1600 (40×40)square cells. HGMF have previously been inoculated with bacterialsuspensions by vacuum filtration and incubated on the differential orselective media of choice.

Because the microbial growth is confined to grid cells of known positionand size on the membrane, the HGMF functions more like an MPN apparatusthan a conventional plate or membrane filter. Peterkin et al. (1987)reported that these HGMFs can be used to propagate and store genomiclibraries when used with a HGMF replicator. One such instrumentreplicates growth from each of the 1600 cells of the ISO-GRID andenables many copies of the master HGMF to be made (Peterkin et al.,1987).

Sharpe et al. (1989) also used ISO-GRID HGMF form QA Laboratories and anautomated HGMF counter (MI-100 Interpreter) and RP-100 Replicator. Theyreported a technique for maintaining and screening many microbialcultures.

Peterkin and colleagues later described a method for screening DNAprobes using the hydrophobic grid-membrane filter (Peterkin et al.,1989). These authors reported methods for effective colony hybridizationdirectly on HGMFs. Previously, poor results had been obtained due to thelow DNA binding capacity of the epoxysulfone polymer on which the HGMFsare printed. However, Peterkin et al. (1989) reported that the bindingof DNA to the surface of the membrane was improved by treating thereplicated and incubated HGMF with polyethyleneimine, a polycation,prior to contact with DNA. Although this early work uses cellular DNAattachment, and has a different objective to the present invention, themethodology described may be readily adapted for Format 3 SBH.

In order to identify useful sequences rapidly, Peterkin et al. (1989)used radiolabeled plasmid DNA from various clones and tested itsspecificity against the DNA on the prepared HGMFs. In this way, DNA fromrecombinant plasmids was rapidly screened by colony hybridizationagainst 100 organisms on HGMF replicates which can be easily andreproducibly prepared.

Manipulation with small (2-3 mm) chips, and parallel execution ofthousands of the reactions. The solution of the invention is to keep thechips and the probes in the corresponding arrays. In one example, chipscontaining 250,000 9-mers are synthesized on a silicon wafer in the formof 8×8 mM plates (15 uM/oligonucleotide, Pease et al., 1994) arrayed in8×12 format (96 chips) with a 1 mM groove in between. Probes are addedeither by multichannel pipette or pin array, one probe on one chip. Toscore all 4000 6-mers, 42 chip arrays have to be used, either usingdifferent ones, or by reusing one set of chip arrays several times.

In the above case, using the earlier nomenclature of the application,F=9; P=6; and F+P=15. Chips may have probes of formula BxNn, where x isa number of specified bases B; and n is a number of non-specified bases,so that x=4 to 10 and n=1 to 4. To achieve more efficient hybridization,and to avoid potential influence of any support oligonucleotides, thespecified bases can be surrounded by unspecified bases, thus representedby a formula such as (N)nBx(N)m.

14.2 Preparation of Support Bound Oligonucleotides

Oligonucleotides, i.e., small nucleic acid segments, may be readilyprepared by, for example, directly synthesizing the oligonucleotide bychemical means, as is commonly practiced using an automatedoligonucleotide synthesizer.

Support bound oligonucleotides may be prepared by any of the methodsknown to those of skill in the art using any suitable support such asglass, polystyrene or Teflon. One strategy is to precisely spotoligonucleotides synthesized by standard synthesizers. Immobilizationcan be achieved using passive adsorption (Inouye & Hondo, 1990); usingUV light (Nagata et al, 1985; Dahlen et al., 1987; Morriey & Collins,1989) or by covalent binding of base modified DNA (Keller et al., 1988;1989); all references being specifically incorporated herein.

Another strategy that may be employed is the use of the strongbiotin-streptavidin interaction as a linker. For example, Broude et al.(1994) describe the use of Biotinylated probes, although these areduplex probes, that are immobilized on streptavidin-coated magneticbeads. Streptavidin-coated beads may be purchased from Dynal, Oslo. Ofcourse, this same linking chemistry is applicable to coating any surfacewith streptavidin. Biotinylated probes may be purchased from varioussources, such as, e.g., Operon Technologies (Alameda, Calif.).

Nunc Laboratories (Naperville, Ill.) is also selling suitable materialthat could be used. Nunc Laboratories have developed a method by whichDNA can be covalently bound to the microwell surface termed Covalink NH.CovaLink NH is a polystyrene surface grafted with secondary amino groups(>NH) that serve as bridge-heads for further covalent coupling. CovaLinkModules may be purchased from Nunc Laboratories. DNA molecules may bebound to CovaLink exclusively at the 5′-end by a phosphoramidate bond,allowing immobilization of more than 1 pmol of DNA (Rasmussen et al.,1991).

Use of CovaLink NH strips for covalent binding of DNA molecules at the5′-end has been described (Rasmussen et al., 1991). In this technology,a phosphoramidate bond is employed (Chu et al., 1983). This isbeneficial as immobilization using only a single covalent bond ispreferred. The phosphoramidate bond joins the DNA to the CovaLink NHsecondary amino groups that are positioned at the end of spacer armscovalently grafted onto the polystyrene surface through a 2 nm longspacer arm. To link an oligonucleotide to CovaLink NH via anphosphoramidate bond, the oligonucleotide terminus must have a 5′-endphosphate group. It is, perhaps, even possible for biotin to becovalently bound to CovaLink and then streptavidin used to bind theprobes.

More specifically, the linkage method includes dissolving DNA in water(7.5 ng/ul) and denaturing for 10 min. at 95° C. and cooling on ice for10 min. Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm₇), is thenadded to a final concentration of 10 mM 1-MeIm₇. A ss DNA solution isthen dispensed into CovaLink NH strips (75 ul/well) standing on ice.

Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC),dissolved in 10 mM 1-MeIm₇, is made fresh and 25 ul added per well. Thestrips are incubated for 5 hours at 50° C. After incubation the stripsare washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3times, then they are soaked with washing solution for 5 min., andfinally they are washed 3 times (where in the washing solution is 0.4 NNaOH, 0.25% SDS heated to 50° C.).

It is contemplated that a further suitable method for use with thepresent invention is that described in PCT Patent Application WO90/03382 (Southern & Maskos), incorporated herein by reference. Thismethod of preparing an oligonucleotide bound to a support involvesattaching a nucleoside 3′-reagent through the phosphate group by acovalent phosphodiester link to aliphatic hydroxyl groups carried by thesupport. The oligonucleotide is then synthesized on the supportednucleoside and protecting groups removed from the syntheticoligonucleotide chain under standard conditions that do not cleave theoligonucleotide from the support. Suitable reagents include nucleosidephosphoramidite and nucleoside hydrogen phosphorate.

An on-chip strategy for the preparation of DNA probe for the preparationof DNA probe arrays may be employed. For example, addressablelaser-activated photodeprotection may be employed in the chemicalsynthesis of oligonucleotides directly on a glass surface, as describedby Fodor et al. (1991), incorporated herein by reference. Probes mayalso be immobilized on nylon supports as described by Van Ness et al.(1991); or linked to Teflon using the method of Duncan & Cavalier(1988); all references being specifically incorporated herein.

To link an oligonucleotide to a nylon support, as described by Van Nesset al. (1991), requires activation of the nylon surface via alkylationand selective activation of the 5′-amine of oligonucleotides withcyanuric chloride.

One particular way to prepare support bound oligonucleotides is toutilize the light-generated synthesis described by Pease et al., (1994,incorporated herein by reference). These authors used currentphotolithographic techniques to generate arrays of immobilizedoligonucleotide probes (DNA chips). These methods, in which light isused to direct the synthesis of oligonucleotide probes in high-density,miniaturized arrays, utilize photolabile 5′-protectedN-acyl-deoxynucleoside phosphoramidites, surface linker chemistry andversatile combinatorial synthesis strategies. A matrix of 256 spatiallydefined oligonucleotide probes may be generated in this manner and thenused in the advantageous Format 3 sequencing, as described herein.

14.3 Preparation of Nucleic Acid Fragments

The nucleic acids to be sequenced may be obtained from any appropriatesource, such as cDNAs, genomic DNA, chromosomal DNA, microdissectedchromosome bands, cosmid or YAC inserts, and RNA, including mRNA withoutany amplification steps. For example, Sambrook et al. (1989) describesthree protocols for the isolation of high molecular weight DNA frommammalian cells (p. 9.14-9.23).

DNA fragments may be prepared as clones in M13, plasmid or lambdavectors and/or prepared directly from genomic DNA or cDNA by PCR orother amplification methods. Samples may be prepared or dispensed inmultiwell plates. About 100-1000 ng of DNA samples may be prepared in2-500 ml of final volume.

The nucleic acids would then be fragmented by any of the methods knownto those of skill in the art including, for example, using restrictionenzymes as described at 9.24-9.28 of Sambrook et al. (1989), shearing byultrasound and NaOH treatment.

Low pressure shearing is also appropriate, as described by Schriefer etal. (1990, incorporated herein by reference). In this method, DNAsamples are passed through a small French pressure cell at a variety oflow to intermediate pressures. A lever device allows controlledapplication of low to intermediate pressures to the cell. The results ofthese studies indicate that low-pressure shearing is a usefulalternative to sonic and enzymatic DNA fragmentation methods.

One particularly suitable way for fragmenting DNA is contemplated to bethat using the two base recognition endonuclease, CviJI, described byFitzgerald et al. (1992). These authors described an approach for therapid fragmentation and fractionation of DNA into particular sizes thatthey contemplated to be suitable for shotgun cloning and sequencing. Thepresent inventor envisions that this will also be particularly usefulfor generating random, but relatively small, fragments of DNA for use inthe present sequencing technology.

The restriction endonuclease CviJI normally cleaves the recognitionsequence PuGCPy between the G and C to leave blunt ends. Atypicalreaction conditions, which alter the specificity of this enzyme(CviJI**), yield a quasi-random distribution of DNA fragments form thesmall molecule pUC19. (2688 base pairs). Fitzgerald et al. (1992)quantitatively evaluated the randomness of this fragmentation strategy,using a CviJI** digest of pUC19 that was size fractionated by a rapidgel filtration method and directly ligated, without end repair, to a lacZ minus M13 cloning vector. Sequence analysis of 76 clones showed thatCviJI** restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, andthat new sequence data is accumulated at a rate consistent with randomfragmentation.

As reported in the literature, advantages of this approach compared tosonication and agarose gel fractionation include: smaller amounts of DNAare required (0.2-0.5 ug instead of 2-5 ug); and fewer steps areinvolved (no preligation, end repair, chemical extraction, or agarosegel electrophoresis and elution are needed). These advantages are alsoproposed to be of use when preparing DNA for sequencing by Format 3.

Irrespective of the manner in which the nucleic acid fragments areobtained or prepared, it is important to denature the DNA to give singlestranded pieces available for hybridization. This is achieved byincubating the DNA solution for 2-5 minutes at 80-90° C. The solution isthen cooled quickly to 2° C. to prevent renaturation of the DNAfragments before they are contacted with the chip. Phosphate groups mustalso be removed from genomic DNA by methods known in the art.

14.4 Preparation of DNA Arrays

Arrays may be prepared by spotting DNA samples on a support such as anylon membrane. Spotting may be performed by using arrays of metal pins(the positions of which correspond to an array of wells in a microtiterplate) to repeated by transfer of about 20 nl of a DNA solution to anylon membrane. By offset printing, a density of dots higher than thedensity of the wells is achieved. One to 25 dots may be accommodated in1 mm², depending on the type of label used. By avoiding spotting in somepreselected number of rows and columns, separate subsets (subarrays) maybe formed. Samples in one subarray may be the same genomic segment ofDNA (or the same gene) from different individuals, or may be different,overlapped genomic clones. Each of the subarrays may represent replicaspotting of the same samples. In one example, a selected gene segmentmay be amplified from 64 patients. For each patient, the amplified genesegment may be in one 96-well plate (all 96 wells containing the samesample). A plate for each of the 64 patients is prepared. By using a96-pin device, all samples may be spotted on one 8×12 cm membrane.Subarrays may contain 64 samples, one from each patient. Where the 96subarrays are identical, the dot span may be 1 mm² and there may be a 1mm space between subarrays.

Another approach is to use membranes or plates (available from NUNC,Naperville, Illinois) which may be partitioned by physical spacers e.g.a plastic grid molded over the membrane, the grid being similar to thesort of membrane applied to the bottom of multiwell plates, orhydrophobic strips. A fixed physical spacer is not preferred for imagingby exposure to flat phosphor-storage screens or x-ray films.

14.5 Sequence Comparisons

Each sequence so obtained was compared to sequences in GenBank using asearch algorithm developed by Applied Biosystems and incorporated intothe INHERIT™ 670 Sequence Analysis System. In this algorithm, PatternSpecification Language (developed by TRW Inc., Los Angeles, Calif.) wasused to determine regions of homology. The three parameters thatdetermine how the sequence comparisons run were window size, windowoffset, and error tolerance. Using a combination of these threeparameters, the DNA database was searched for sequences containingregions of homology to the query sequence, and the appropriate sequenceswere scored with an initial value. Subsequently, these homologousregions were examined using dot matrix homology plots to distinguishregions of homology from chance matches. Smith-Waterman alignments wereused to display the results of the homology search. Peptide and proteinsequence homologies were ascertained using the INHERIT™ 670 SequenceAnalysis System in a way similar to that used in DNA sequencehomologies. Pattern Specification Language and parameter windows wereused to search protein databases for sequences containing regions ofhomology that were scored with an initial value. Dot-matrix homologyplots were examined to distinguish regions of significant homology fromchance matches.

Alternatively, BLAST, which stands for Basic Local Alignment SearchTool, is used to search for local sequence alignments (Altschul S F(1993) J Mol Evol 36:290-300; Altschul, SF et al (1990) J Mol Biol215:403-10). BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs. Whereas it is ideal for matcheswhich do not contain gaps, it is inappropriate for performingmotif-style searching. The fundamental unit of BLAST algorithm output isthe High-scoring Segment Pair (HSP). An HSP consists of two sequencefragments of arbitrary but equal lengths whose alignment is locallymaximal and for which the alignment score meets or exceeds a thresholdor cutoff score set by the user. The BLAST approach is to look for HSPsbetween a query sequence and a database sequence, to evaluate thestatistical significance of any matches found, and to report only thosematches which satisfy the user-selected threshold of significance. Theparameter E establishes the statistically significant threshold forreporting database sequence matches. E is interpreted as the upper boundof the expected frequency of chance occurrence of an HSP (or set ofHSPs) within the context of the entire database search. Any databasesequence whose match satisfies E is reported in the program output.

The present invention is illustrated in the following examples. Example1 addresses the identification of a novel interleukin-1 receptorantagonist. Example 2 addresses expression of studies of SEQ ID NO: 2 inhuman tissues using semi-quantitative PCR. Example 3 addresseschromosomal localization using SEQ ID NO: 2. Example 4 addresses thedetermination of intron/exon structure of locus corresponding to SEQ IDNO: 2. Example 5 addresses the identification of Interleukin-1 receptorbinding domain and receptor binding assays. Example 6 and 7 address thedetermination of the nucleotide sequence of IL-1 Hy1. Example 8 and 11address the isolation and mapping of the IL-1 Hy1 genomic clone. Example9 addresses IL-1 Hy1 expression in E. coli. Example 10 addresses thebiological uses of IL-1 Hy1. Example 12 addresses recombinant proteinexpression and purification of IL-1 Hy1 polypeptide. Example 13addresses binding of interleukin-1 Hy1 receptor antagonist to the IL-1receptor. Example 14 addresses the confirmation of IL-1 Hy1 IL-1antagonist activity. Example 15 addresses the inhibition of IL-1βinduced IL-6 production. Example 16 addresses the determination of IL-1Hy1 molecular mass. Example 17 addresses IL-1 Hy1 expression inactivated THP-1 cells. Example 18 addresses the detection of IL-1 Hy1protein expression in human tissues by immunohistochemistry. Example 19addresses detection of IL-1 Hy1 by in situ hybridization with a DNAprobe. Example 20 addresses detection of IL-1 Hy1 by in situhybridization with a riboprobe. Example 21 describes induction of IL-13production by IL-1 Hy1. Example 22 relates to inhibition of IL-18activity by IL-1 Hy1 as measured by induction of interferon-gammaexpression.

Upon consideration of the present disclosure, one of skill in the artwill appreciate that many other embodiments and variations may be madein the scope of the present invention. Accordingly, it is intended thatthe broader aspects of the present invention not be limited to thedisclosure of the following examples.

7.0. EXAMPLES 7.1 Example 1 A Novel Interleukin-1 Receptor AntagonistObtained from a cDNA Library of Fetal Liver-Spleen

A plurality of novel nucleic acids were obtained from the b²HFLS20W cDNAlibrary prepared from human fetal liver-spleen tissue, as described inBonaldo et al., Genome Res. 6:791-806 (1996), using standard PCR, SBHsequence signature analysis and Sanger sequencing techniques. Theinserts of the library were amplified with PCR using primers specificfor vector sequences which flank the inserts. These samples were spottedonto nylon membranes and interrogated with oligonucleotide probes togive sequence signatures. The clones were clustered into groups ofsimilar or identical sequences, and single representative clones wereselected from each group for gel sequencing. The 5′ sequence of theamplified inserts was then deduced using the reverse M13 sequencingprimer in a typical Sanger sequencing protocol. PCR products werepurified and subjected to flourescent dye terminator cycle sequencing.Single pass gel sequencing was done using a 377 Applied Biosystems (ABI)sequencer. Two (2) of these inserts have been identified as novelsequences not previously obtained from this library, and not previouslyreported in public databases. These sequences are shown in FIG. 2 as SEQID NO: 1 and 2. The polypeptide sequences corresponding to these nucleicacid sequences are shown in FIG. 3 as SEQ ID NO: 3. These amino acidsequences have striking homology to Interleukin-1 receptor antagonist.

7.2 Example 2 Expression Study Using SEQ ID NO: 2

To study the role of SEQ ID NO: 2 in the regulation of the inflammatoryresponse, gene expression was analyzed using a semi-quantitativepolymerase chain reaction-based technique. cDNA libraries were used assources of expressed genes from tissues of interest (three leukocytepreparations [two stimulated and one unstimulated], heart, lung, spleen,placenta, testes, fetal liver, adult liver, bone marrow, lymph node,macrophages, endothelial cells, fetal skin, and umbilical cord). Genespecific primers were used to amplify portions of the SEQ ID NO: 2sequence (corresponding to bases 105-772 and 161-690, as numbered fromthe 5′ end of SEQ ID NO: 2) from the samples. Amplified products wereseparated on an agarose gel, transferred and chemically linked to anylon filter. The filter was then hybridized with a radioactivelylabeled (³³Palpha-dCTP) double-stranded probe generated from thefull-length SEQ ID NO: 2 sequence using a Klenow polymerase, randomprime method. The filters were washed (high stringency) and used toexpose a phosphorimaging screen for several hours. Bands indicated thepresence of cDNA including SEQ ID NO: 2 sequences in a specific library,and thus mRNA expression in the corresponding cell type or tissue.

SEQ ID NO: 2 was expressed in a very limited set of human tissues. Ofthe 16 human tissues tested, fetal skin and umbilical cord were the onlysamples that provided a signal, indicating that expression of SEQ ID NO:2 is tightly regulated. Expression of both IL-1 Ra are tightlyrestricted to a subset of tissues and cell types; both constitutivelyexpressed in skin but not present in other tissues or cell types withoutstimulation Thus, the expression pattern of SEQ ID NO: 2 parallels thatof the IL-1 Ra genes, indicating that the novel cytokine encoded by SEQID NO: 2 plays a role in the regulation of the inflammatory response.

7.3 Example 3 Chromosomal Localization Study Using SEQ ID NO: 2

Chromosome mapping technologies allow investigators to link genes tospecific regions of chromosomes. Chromosomal mapping was performed withthe Stanford G3 Radiation Hybrid Panel (Research Genetics). The panelwas screened with gene-specific primers (5′ primer:CCCCACTGGATGGTGCTACTG; (SEQ ID NO: 15), 3′ primer:GGGAAGAGATAGGAAAGGTAG) (SEQ ID NO: 16)that generated a sequence tag site(STS), and the results of the PCR screening were submitted to theStanford Radiation Hybrid mapping email server at the Stanford HumanGenome Center (SHGC). The gene position on the radiation hybridframework map was provided by linking the STS corresponding to SEQ IDNO: 2 with the SHGC marker with best linkage.

The results indicated that SEQ ID NO: 2 is located on the long arm ofchromosome 2. The STS was linked to the marker SHGC-7020 with a LOD (logof the odds) score of 12.25 and cR-1000 of 5, indicating that the STSwas within 120 kb (kilobases) of this marker. SHGC-7020 is in turnlocated within 120 kb of the IL-1 Ra gene. Thus, the STS correspondingto SEQ ID NO: 2 is located within about 240 kilobases of the IL-1 Ragene and could be in close proximity to the IL-1 Ra gene.

Gene family members are often linked to specific regions of chromosomesowing to intrachromosomal gene duplication events that give rise tomultimember gene families during the process of evolution. Theinterleukin-1 gene family has been mapped to chromosome 2. Morespecifically, all of the interleukin 1 genes (IL-1a, IL-1b) and thereceptors (IL-1 RI and IL-1 RII), as well as the receptor antagonistIL-1 Ra, have been found to be situated on the long arm of chromosome 2.The identification of SEQ ID NO: 2 sequences in this same regionestablishes the physical linkage of SEQ ID NO: 2 to the interleukin-1locus which evidences that the cytokine encoded by SEQ ID NO: 2functions as a modulator of the inflammatory response.

7.4 Example 4 Intron/Exon Structure of the Locus Corresponding to SEQ IDNO: 2

Members within gene families often maintain genomic organization byvirtue of the fact that they arose from a common ancestral precursor.The intron/exon structure of IL-1a, IL-1b and IL-1 Ra have been highlyconserved, demonstrating that they probably arose from a commonprecursor gene. We have isolated a bacterial artificial chromosome (BAC)containing SEQ ID NO: 2. Using gene-specific primers we have sequencedin the 5′ direction of the clone, delineating coding sequence (identicalto SEQ ID NO: 2) and intronic sequence. The intron/exon structure isidentical between IL-1 Ra and the BAC fragment containing SEQ ID NO: 2,providing evidence that these two sequences are members of the samefamily and were generated from a common gene precursor.

7.5 Example 5 Interleukin-1 Receptor Binding Domain and Interleukin-1Receptor Assay

The receptor binding region of both IL-1β and IL-1 Ra have been mappedan 18 amino acid region in the carboxy terminal half of the proteins(i.e., residues 88-105 of IL-1β) by site-directed mutagenesis andprotein modification studies. An amino acid alignment of SEQ ID NO: 3with both IL-1β and IL-1 Ra demonstrates that SEQ ID NO: 3 contains areceptor binding region. SEQ ID NO: 3 is 39% identical to IL-1 Ra and22% identical (39% conserved) with IL-1β in this region. In comparison,IL-1 Ra, which is known to bind to the IL-1 receptor, is 28% identicaland 50% conserved with IL-1β in this region. The remarkable similaritybetween this region of SEQ ID NO: 3 and the receptor binding regions ofboth IL1β and IL-1 Ra indicates that SEQ ID NO: 3 also contains an IL-1receptor binding region. The alignment of all three proteins in thereceptor binding region is shown in FIG. 4.

Because SEQ ID NO: 3 contains a IL-1 receptor binding region, SEQ ID NO:3 and truncated forms of SEQ ID NO: 3 that include the receptor bindingregion are useful as reagents to identify cells and tissues expressingIL-1 receptors. The IL-1 receptor binding assay described in Hannum etal. Nature 343:336-340 (1990) is used. Briefly, highly radioactiverecombinant SEQ ID NO: 3 is prepared by growing E. coli expressing SEQID NO: 3 on M9 medium containing [³⁵S] sulphate and purifying thelabeled SEQ ID NO: 3 by chromatography on a Mono-S column. The labeledSEQ ID NO: 3 is incubated with the cells or tissue under standard IL-1binding assay conditions, and [³⁵S] binding. Significant [³⁵S] bindingindicates the presence of IL-1 receptors.

7.6 Example 6 Determination of a Nucleotide Sequence Encoding a155-Amino Acid Protein with Sequence Homology to Human Interleukin-1beta and Human Interleukin-1 Receptor Antagonist

The nucleotide sequence presented in FIG. 5, and labeled SEQ ID NO: 4,encodes the translated amino acid sequence SEQ iD NO: 5, which ispresented in FIG. 6. The extended nucleotide sequence was obtained byisolating PCR products generated from pools of clones from a fetal skincDNA library. In short, a fetal skin cDNA library was plated onampicillin containing plates in pools of about 40,000 colonies. Thecolonies were recovered into LB medium and PCR was used to detect poolswhich contained SEQ ID NO: 2 (FIG. 2). Two pools were identified. PCRusing vector- and gene-specific primers amplified the 5′ portion of thecDNA. Nested primers were used to generate sequence from the twoamplified products. Laser gene™ software was used to edit and “contig”the partial sequences into a full length sequence.

SEQ ID NO: 4 encodes a protein of 155 amino acids, lacking a typicalhydrophobic leader peptide, suggesting that this protein is retained asa cytoplasmic molecule, similar to the cytoplasmic isoform of the humanIL-1 Ra gene product. FIG. 7 presents an amino acid alignment of SEQ IDNO: 5 with the cytoplasmic form of human IL-1 Ra (labeled“HUMIL1RASIC”). The alignment reveals a high degree of homology betweenthe two; 48% of the amino acids were identical and 54% representconservative amino acid substitutions. Three residues have been provento be critical for receptor activation by IL-1β (marked with asterisksin FIG. 7). The corresponding residues in IL-1 Ra differ, conferringIL-1 Ra's antagonistic activity. These residues are R21, W23, and K152for IL-1 Ra, and T9, R11, and D145 for the mature IL-1β. SEQ ID NO: 5possesses a combination of both residues, R10 (identical to thecorresponding residue in IL-1 Ra) and K12 and D148 (similar andidentical, respectively, to the corresponding residues in IL-1β). Theoverall homology and agonist/antagonist site-specific sequencehomologies clearly define SEQ ID NO: 5 as a protein modulator of theinflammatory response.

7.7 Example 7 Three Prime Extension of SEQ ID NO: 4

SEQ ID NO: 6 (FIG. 8) is an extension of the 3′ end of the nucleic acidsequence of SEQ ID NO: 4. SEQ ID NO: 6 was obtained from a fetal skincDNA library as described above for SEQ ID NO: 4.

7.8 Example 8 Isolation and Mapping of a Genomic Clone Corresponding toSEQ ID NOS: 1, 2, and 4

A human BAC genomic library (Research Genetics) was screened withgene-specific primers (273-D, 5′-CCCCACTGGATGGTGCTACTG-3′ (SEQ ID NO:15) which hybridizes at position 4533 to 4553 in the genomic sequenceand 273-E, 5′-GGGAAGAGATAGGAAAGGTAG-3′ (SEQ ID NO: 16) which hybridizesat position 4849 to 4869) using a PCR based assay. Briefly, the genespecific primers were used to amplify BAC DNAs as templates usingstandard PCR conditions. BACs that produce a fragment of DNAcorresponding to the predicted size were pursued. BAC393-16 was isolatedand its DNA sequenced with gene-specific primers derived from SEQ ID NO2. The sequence (16403 bases in length) is shown in FIG. 9. The IL-1 Racoding sequence was found to be distributed over 5 exons. The splicedonor and acceptor sites are shown below, along with intron and exonsizes.

Size Exon Exon Splice Donor Intron Splice Acceptor Size 1 TCTGAGgtatgc 172 bp aaatagGGGAGT 2 (SEQ ID NO:17) (SEQ ID NO:18) (66 bp) 2CTTCCGgtgagt 1396 bp tttcagAATGAA 3 (SEQ ID NO:19) (SEQ ID NO:18) (87bp) 3 TTAAAGGTTGGT 1189 bp ccacagGTGAAG 4 (SEQ ID NO:21) (SEQ ID NO:22)(120 bp) 4 CTAGAGgtgaga  204 bp cggcagCCAGTG (SEQ ID NO:23) (SEQ IDNO:24)

7.9 Example 9 Expression of SEQ ID NO: 3 in E. coli

SEQ ID NO: 3 was expressed in E. coli by subcloning the entire codingregion of the SEQ ID NO: 2 into a prokaryotic expression vector. Theexpression vector (pQE16) used was from the QlAexpression prokaryoticprotein expression system (Qiagen). The features of this vector thatmake it useful for protein expression include: an efficient promoter(phage T5) to drive transcription; expression control provided by thelac operator system, which can be induced by addition of IPTG(isopropyl-β-D-thiogalactopyranoside), and an encoded His₆ tag. Thelatter is a stretch of 6 histidine amino acid residues which can bindvery tightly to a nickel atom. The vector can be used to express arecombinant protein with a His₆ tag fused to its carboxyl terminus,allowing rapid and efficient purification using Ni-coupled affinitycolumns.

The coding sequence of SEQ ID NO: 2 (including the start codon butexcluding the stop codon) was amplified using the PCR reaction primersAP1 (5′ gaagatctatggtcctgagtggggccctg-3′) (SEQ ID NO: 25) and AP2 (5′gaagatctgtcacactgctggaagtagaa-3′) (SEQ ID NO: 26). Both primers have BglII restriction sites incorporated at the 5′ ends for cloning purposes.The PCR fragment obtained upon amplification was restricted with Bgl IIto generate staggered ends for ligating the insert into the vector. ThepQE16 plasmid was digested with Bgl II and BamHI to remove a segmentcontaining the dihydrofolate reductase coding sequence and also togenerate the staggered ends compatible with those on the PCR fragment.The PCR fragment was ligated into the digested pQE 16 vector to producep16BB-273. The ligation was transformed by electroporation intoelectrocompetant E. coli cells (strain M15[pREP4] from Qiagen), and thetransformed cells were plated on ampicillin-containing plates. Colonieswere screened for the correct insert in the proper orientation using aPCR reaction employing a gene-specific primer and a vector-specificprimer. Positives were then sequenced to ensure correct orientation andsequence. To express SEQ ID NO: 3, a colony containing a correctrecombinant clone was inoculated into L-Broth containing 100 μg/ml ofampicillin, 25 μg/ml of kanamycin, and the culture was allowed to growovernight at 37° C. The saturated culture was then diluted 20-fold inthe same medium and allowed to grow to an optical density at 600 nm of0.5. At this point, IPTG was added to a final concentration of 1 mM toinduce protein expression. The culture was allowed to grow for 5 morehours, and then the cells were harvested by centrifugation at 3000×g for15 minutes.

The resultant pellet was lysed using a mild, nonionic detergent in 20 mMTris HCl (pH 7.5) (B-PER™ Reagent from Pierce), or by sonication untilthe turbid cell suspension turned translucent. The lysate obtained wasfurther purified using a nickel containing column (Ni-NTA spin columnfrom Qiagen) under non-denaturing conditions. Briefly, the lysate wasbrought up to 300 mM NaCl and 10 mM imidazole and was centrifuged at700×g through the spin column to allow the His-tagged recombinantprotein to bind to the nickel column. The column was then washed twicewith Wash Buffer (50 mM NaH₂PO₄, pH 8.0; 300 mM NaCl; 20 mM imidazole)and was eluted with Elution Buffer (50 mM NaH₂PO₄, ph 8.0; 300 mM NaCl;250 mM imidazole). All the above procedures were performed at 4° C. Thepurified protein was checked with SDS-PAGE. A strong single band wasobserved, indicating a molecular weight of 16 kD, which is consistentwith the predicted size of SEQ ID NO: 3.

7.10 Example 10 Use of SEQ ID NOS: 3 and 5

7.10.1 Medical Imaging

The novel Interleukin-1 receptor antagonist polypeptides of theinvention are useful in medical imaging, e.g., imaging the site ofinfection, inflammation, and other sites having Interleukin-1 receptorantagonist receptor molecules. See, e.g., Kunkel et al., U.S. Pat. No.5,413,778. Such methods involve chemical attachment of a labeling agent,administration of the labeled Interleukin-1 receptor antagonistpolypeptide to a subject in a pharmaceutically acceptable carrier, andimaging the labeled Interleukin-1 receptor antagonist polypeptide invivo at the target site.

7.10.2 Pancreatitis

Acute edematous, necrotizing pancreatitis is induced in adult male Swissmice weighing more than 35 grams using caerulein—an analog ofcholecystokinin. Mice are divided into four groups with three of thegroups receiving caerulein 50 mu g/kg by intraperitoneal (IP) injectionin four doses over three hours as previously described. (Murayama etal., Arch Surg 1990;125:1570-1572; Tani et al., International JPancreatology 1987;2:337-348; Schoenberg et al., Free Radical Biology &Medicine 1992;12:515-522; Heath et al., Pancreas 1993;66:41-45; Salujaet al., Amer Physiological Society 1985: G702-G710; Manso et al.,Digestive Disease and Sciences 1992;37:364-368).

Group 1 is a control group (n-9) which received only IP salineinjections. Group 2 (n=12) is an untreated disease control. Group 3(n=12) received three injections (10 mg/kg/hr) starting one hour priorto induction of pancreatitis. Group 4 (n=12) received three injections(10 mg/kg/hr) starting one hour after induction of pancreatitis.

After a suitable time period, all animals are euthanized, the bloodcollected, and the pancreata surgically excised and weighed. Serum isassayed for amylase, lipase, IL-6, and TNF levels. Each pancreas isfixed, stained, and graded histologically in a blinded fashion forinterstitial edema, granulocyte infiltration, acinar vacuolization, andacinar cell. Additionally, serum levels of Interleukin-1 receptorantagonist are determined, therefore allowing comparisons betweendosage, serum level, systemic cytokine response, and degree ofpancreatic damage.

lnterleukin-6, Interleukin-1, Interleukin-1 receptor antagonist, and TNFare measured by commercially available ELISA kits (Genzyme Corp.,Boston, Mass.). All specimens are run in triplicate. Serum levels ofamylase and lipase are measured on a Kodak Ectachem 700 automatedanalyzer (Eastman Kodak Company, Rochester, N.Y.).

Histologic slides are prepared as is known in the art after rapidexcision and subsequent fixation in 10% formalin. The tissues areparaffin embedded as is known in the art and then stained withHematoxylin and Eosin in a standard fashion. These slides are examinedand graded in a blinded fashion by a board certified pathologist.

7.10 3 Inhibition of Interleukin-1 Induced Cell Proliferation

Murine D10 T cells are obtained from the American Type CultureCollection (Rockville, Md.). Cells are maintained in Dulbecco's modifiedEagle medium and Ham's F-12 medium (1:1) containing 10 mM HEPES buffer(pH 7.4) and 10% fetal bovine serum. All tissue culture reagentscontained less than 0.25 ng/mL endotoxin as measured by the limulusamebocyte assay.

Murine D10 cells, an Interleukin-1 dependent T-cell line, are used tomeasure Interleukin-1 mitogenic activity. Cell proliferation in thepresent of Interleukin-1 with and without the IL-1 Hy2 polypeptides ofthe invention is assessed by incorporation of (³H) thymidine aspreviously described (Bakouche, O., et al. J. Immunol. 138:4249-4255,1987). In a preferred embodiment, antagonists and agonist of the IL-1Hy2 polypeptides of the invention are identified in this assay by addingthe candidate compounds with the Interleukin-1 and IL-1 Hy2 polypeptidesof the invention and measuring the change in cell proliferation causedby the candidate compound.

7.10.4 Inhibition of Interleukin-1 Induced Cell Cytoxicity

Inhibition of Interleukin-1-induced cytotoxicity is studied using anappropriate cell line, such as, for example, A375 tumor cells plated ata density of 6000 cells per well in 96-well microliter plates. Afterovernight attachment, Interleukin-1 (3-300 ng/mL) is added in thepresence or absence of NAA or NMA. After cells are incubated for 3 days,(³ H) thymidine is added (1 mu Ci per well) for an additional 2 hours.Cells are harvested onto glass fiber disks (PHD Cell Harvested;Cambridge Technology, Inc., Watertown, Mass.) Disks are air driedovernight, and radioactivity is determined with a Model 1900TRScintillation Counter (Packard Instrument Division, Downers Grove, Ill.)

7.10.5 Induction of Nitrite Synthesis in Smooth Muscle Cells

Aortic smooth muscle cells are cultured by explanting segments of themedial layer of aortas from adult male Fischer 344 rats. Aortas areremoved aseptically and freed of adventitial and endothelial cells byscraping both the luminal and abluminal surfaces. Medial fragments areallowed to attach to Primaria 25-cm² tissue culture flasks(Becton-Dickinson, Lincoln Park, N.J.) which are kept moist with growthmedium until cells emerged. Cultures are fed twice weekly with medium199 containing 10% fetal bovine serum, 25 mM HEPES buffer (pH 7.4), 2 mML-glutamine, 40 mu g/mL endothelial cell growth supplement (BiomedicalTechnologies, Inc., Stoughton, Mass.) and 10 mu g/ml gentamicin (GIBCOBRL, Grand Island, N.Y.). When primary cultures become confluent, theyare passaged by trypsinization, and explants are discarded. For thesestudies, cells from passages 12-14 are seeded at 20,000 per well in96-well plates and are used at confluence (60,000-80,000 cells perwell). The cells exhibit the classic smooth muscle cell phenotype withhill and valley morphology, and they stain positively for smooth muscleactin.

Rat aortic smooth muscle cells are incubated with RPMI-1640 mediumcontaining 10% bovine calf serum, 25 mM HEPES buffer 7.4), 2 mMglutamine, 80 U/mL penicillin, 80 mu g/mL streptomycin, 2 mu g/mLfungizone, and Interleukin-1, IFN-gamma, and various inhibitors. At thedesired times, nitrite concentration in the culture medium is measuredusing the standard Griess assay (Green, L., et al. Anal. Biochem.126:131-138, 1982) adapted to a 96-well micro titer plate reader (Gross,S. S., et al. Biochem. Biophys. Res. Commun. 178:823-829, 1991). Thus,100 uL of Griess reagent (0.5% sulfanilic acid, 0.05%naphthalenediamine, and 2.5% phosphoric acid) is added to an equalvolume of culture medium, and the OD sub 550 is measured and related tonitrite concentration by reference to a standard curve. The backgroundOD sub 550 of medium incubated in the absence of cells is subtractedfrom experimental values.

Rat aortic smooth muscle cells are incubated with RPMI-1640 mediumcontaining 10% bovine calf serum, 25 mM HEPES buffer (pH 7.4), 2 mMglutamine, 80 mu g/mL penicillin, 80 mu g/mL steptomycin, 2 mu g/mLfungizone, 30 mu g/mL lipopolysaccharide (Escherichia coli 0111:B4), and50 U/mL IFN-γ. Cells are harvested after 24 hours, and cytosol isprepared (Gross, S. S., et al. Biochem. Biophys. Res. Commun.178:823-829, 1991). Cytosolic NO synthase activity is assayed by the Fe2+-myoglobin method described previously (Gross, S. S., et al. Biochem.Biophys. Res. Commun. 178:823-829, 1991).

7.10.6 Alloreactivity Determined by Lymph Node Weight Gain

Experiments are conducted to show that systemic administration of theInterleukin-1 receptor antagonists polypeptides of the inventionsuppress a localized, T cell-dependent, immune response to alloantigenpresented by allogeneic cells. Mice are injected in the footpad withirradiated, allogeneic spleen cells. The mice are then injected in thecontralateral footpad with irradiated, syngeneic spleen cells. Analloreactive response (marked by proliferation of lymphocytes andinflammation) occurs in the footpad receiving the allogeneic cells,which can be measured by determining the increase in size and weight ofthe popliteal lymph node draining the site of antigen depositionrelative to controls or by an increase in cellularity.

Specific pathogen free 8-12 week old BALB/c (H-2 sup d) and C57BL/6 (H-2sup b) mice (Jackson Laboratory, Bar Harbor, Me.) are used in thisexperiment. 48 BALB/c mice are divided into 16 groups, each having 3mice (unless otherwise indicated). Each group of mice received adifferent mode of treatment. On day 0 the left footpads of all mice areinjected intracutaneously with 107 irradiated (2500R), allogeneic spleencells from C57BL/6 mice in 50 μL of RPMI-1640 (Gibco) as antigen and theright contralateral footpads of the same mice are injected with 10 sup 7irradiated (2500R), syngeneic spleen cells from BALB/c mice.

Seven days after antigen administration, the mice are sacrificed and thepopliteal lymph nodes (PLN) are removed from the right and leftpopliteal fossa by surgical dissection. Lymph nodes are weighed and theresults expressed as the difference (DELTA) in weight (mg) of the lymphnode draining the site of allogeneic cell injection and the weight ofthe node draining the syngeneic cell injection site. Lymph nodesdraining the syngeneic cell injection site weighed approximately 1 mg,regardless of whether they are obtained from mice treated with MSA orInterleukin-1 receptor antagonist polypeptides of the invention, and didnot differ significantly in weight from nodes obtained from mice givenno cell injection.

7.10.7 Suppression of Organ Graft Rejection In Vivo

Neonatal C57BL/6 (H-2 sup b) hearts are transplanted into the ear pinnaeof adult BALB/c (H-2 sup d) recipients utilizing the method of Fulmer etal., Am. J. Anat. 113:273, 1963, modified as described by Trager et al.,Transplantation 47:587, 1989, and Van Buren et al., Transplant. Proc.15:2967, 1983. Survival of the transplanted hearts is assessed byvisually inspecting the grafts for pulsatile activity. Pulsatileactivity is determined by examining the ear-heart grafts of anesthetizedrecipients under a dissecting microscope with soft reflected lightbeginning on day 5 or 6 post transplant. The time of graft rejection isdefined as the day after transplantation on which contractile activityceases.

Recipient mice are transplanted on day 0 and injected with eitherinterleukin-1 receptor antagonist polypeptides of the invention plus MSA(mouse serum albumin, 100 ng) or with MSA alone on days 0 through 6,alternating i.p. and s.c. routes. In a second heart transplantexperiment, the mice are injected with MSA alone on days 0 through 2,i.p. route only.

7.10.8 Suppression of Inflammatory Arthritis

20 rats are divided into 4 groups, designated Groups G-J, each having 5rats. All rats are immunized by subcutaneous injection. On day 21following immunization with nBSA, an inflammatory arthritis response iselicited. On the same day, a negative control group is injected with a0.2 ml volume of saline. Groups are injected with increasing amounts ofInterleukin-1 receptor antagonist polypeptides of the invention.Interleukin-1 is injected in one group as a positive control. Thediameter of the largest region of the treated joints is measured using acaliper on days 2, 4, 6 and 8 relative to day 0 intra-articularinjection of antigen.

7.11 Example 11 Isolation and Mapping of a Genomic Clone Correspondingto SEQ ID NO: 6

FIGS. 10A-C show a genomic clone (SEQ ID NO: 8) which is an extension ofthe genomic sequence presented in FIGS. 9A-C (SEQ ID NO: 7). SEQ ID NO:8 includes the extended sequence (three prime untranslated) shown in SEQID NO: 6. The isolation of this genomic clone (SEQ ID NO: 8 ) was thesame as described in Example 8. The sequence is 7,605 nucleotides inlength) is shown in FIGS. 10A-C. The organization of the exons for thisclone is described below.

Nucleotide Intronic/Exonic Exon Range of Exon Sequeces*** Exon 1E*1308-1383 ctgtagGCCTGG - - - AAAAAGgtaagg(SEQ ID NOS: 27 & 28) Exon 1M**1780-1890 cctcagGTCCTG - - - TCTGAGgtatgc(SEQ ID NOS: 29 & 17) Exon 22061-2116 aaatagGGGAGT - - - CTTCCGgtgagt(SEQ ID NOS: 18 & 19) Exon 33504-3589 tttcagAATGAA - - - TTAAAGgttggt(SEQ ID NOS: 20 & 21) Exon 44777-4905 ccacagGTGAAG - - - CTAGAGgtgaga(SEQ ID NOS: 22 & 23) Exon 55107-7395 cggcagCCAGTG - - - AAAGAG(SEQ ID NO: 24) *Exon 1E, exon 1 inepidermoid carcinoma cell line A431 **Exon 1M, exon 1 in activatedmacrophage cell line THP1 ***Intronic sequences shown in lowercase,exonic sequences shown in uppercase.

7.12 Example 12 Recombinant Protein Expression and Purification

An expression vector placing the Interleukin-1 Receptor Antagonistcoding sequence (SEQ ID NO: 3) without the N terminal hydrophobicsequence was placed after the His₆ tag (a stretch of 6 histidine aminoacid residues) and express tags (antibody tag) in the pRSET vector(conferring ampicillin resistance). The His₆ tag fused to its aminoterminus of a recombinant protein allows rapid and efficientpurification using Ni-coupled affinity columns.

This construct was transformed into E. coli (BL-21 with the pLYS plasmidconferring chloramphenicol resistance) and expression was induced usingIPTG (1 mM final). Bacterial pellet was suspended in 50 mM Tris pH.7.5.Sonic dismembrator was employed to lyse cells in the presence of 1 mg/mlof lysozyme (10 ml of buffer per gram of wet cells). Cell debris wasremoved by centrifugation. Imidazole was added at 10 mM finalconcentration with 100 mM NaCl. The extract was loaded on a Nickelchelate column. The protein was eluted off the column with a lineargradient of imidazole from 50 mM to 300 mM. After the peak wascollected, it was desalted on a sephadex G-25 column into 20 mM sodiumphosphate buffer. Protein was loaded onto a Q-sepharose column andeluted off the gradient from 0-350 mM NaCl. The final recombinantprotein was filter sterilized and stored at −20° C.

7.13 Example 13 Binding of the Interleukin-1 Hy1 Receptor Antagonist tothe Interleukin-1 Receptor

A cell binding assay was carried out to demonstrate thatInterleukin-1Hy1 Receptor Antagonist of the invention binds to theInterleukin-1 receptor. Briefly, cell binding of the recombinant protein(see Example 12) with and without the presence of 100 fold greateramounts of non tagged Interleukin-1 Beta (IL-1β) ligand was analyzed byusing fluorescent antibodies specific for the express tag in thelnterleukin-1 Hy1 Receptor Antagonist recombinant protein on thefluorescent activated cell sorter (FACS). In each reaction, 10⁶ cellsNHDF (normal human dermal fibroblasts) were resuspended in 100 μl ofFACS buffer (distilled PBS and 3% calf serum and 0.01% azide). Cellbinding was done by adding 5 nM recombinant Interleukin-1 Hy1 ReceptorAntagonist in 100 μl cell suspension and as a competition in onereaction, 500 nM of recombinant IL-1β was also added. The cells wereincubated on ice for 1 hr. The cells were pelleted, 200 μl of 0.2 mM BS3(crosslinker) was added, and the cells were kept on ice for 30 min.Next, 10 μl 1 M Tris pH 7.5 was added and the cells were incubated for15 minutes on ice. The cells were pelleted, washed 1 time in FACSbuffer, resuspended in 100 μl volume of FACS buffer and 2 μl primaryantibody (anti-express tag antibody 1 mg/ml) was added, and incubated onice for 30 min. The cells were pelleted, washed with FACS buffer, andresuspended in FACS buffer (100 μl volume). The secondary antibody(phycoerythrin conjugated) 2 ul of anti-mouse Ig (1 mg/ml) was added andthe cells were incubated for 30 minutes on ice. The cells were againpelleted, washed two times with FACS buffer, resuspended in 0.5 ml FACSbuffer and analyzed on FACS. A shift in the fluorescence was observed inthe cells treated with the recombinant tagged Interleukin-1Hy1 ReceptorAntagonist. This binding was shown to be specific, as is was competedoff with the non-tagged IL-1β protein. These results indicate binding ofthe Interleukin-1 Hy1 Receptor Antagonist protein of the invention tothe IL-1 receptor.

The assay was repeated using unlabeled IL-1 Hy1 Receptor Antagonist(IL-1 Hy1 RA) as the ligand. A shift in fluorescence was observed forthe cells treated with the recombinant tagged IL-1 Hy1. This binding wasspecific, as binding was decreased in the presence of the non-taggedIL-1 Hy1 RA protein. These results confirmed binding of the IL-1 Hy1protein of the invention to the IL-1 receptor.

7.14 Example 14 Confirmation of IL-1 Antagonist Activity

IL-1 antagonist activity was determined using a prostaglandin E₂ (PGE₂)based assay as follows. NHDF cells were plated at 2×10⁴ cells per wellin a 96-well plate 24 hours before the assay. The cells were thentreated with 25 pg/ml recombinant human IL-1β (R&D Systems) for 16 hoursat 37° C. in a 5% CO₂ incubator. To study the inhibition of IL-1βstimulated PGE₂ release by IL-1Hy1, the cells were pretreated withdifferent amounts of IL-1Hy1 for two hours before the addition of IL-1.The supernatants were then collected and cell debris was removed bycentrifugation. The amount of PGE₂ in the supernatants was determined byELISA using the PGE₂ assay system (R&D Systems) according to themanufacturer's directions.

When NHDF cells were treated with IL-1Hy1 alone, no PGE₂ activity wasmeasured. To determine whether IL-1Hy1 could inhibit IL-1β induced PGE₂production, the cells were pretreated with different concentrations ofIL-1Hy1 before stimulation with IL-1β. IL-1Hy1 was able to inhibit IL-1βinduced PGE₂ production in a dose dependent fashion as shown in FIG. 11.These results indicate that IL-1Hy1 acts in a manner similar to IL-1rain vitro, indicating that they have overlapping functions.

The assay may alternatively be conducted with CCD 1098 human fibroblastcells (ATCC Accession No. CRL2127) as follows. Approximately 2×10⁴ cellsper well were seeded in 96 well plate the day before cell stimulation.On the day of stimulation, cells were washed once with fresh medium andreplated with 200 μl fresh medium in each well. The cells werestimulated with IL-1β (at a final concentration of 1 ng/ml) or with thecytokine of interest alone. To test PGE₂ inhibition, differentconcentrations of IL-1 Hy1 or IL-1ra were added to the wells togetherwith IL-1β. After 16 hours of cell stimulation, the culture plate wasspun for 5 minutes at 4000 rpm to remove cell debris. 100 μl supernatantwas removed to test for the presence of PGE₂ using PGE₂ assay kitmanufactured by R & D system. The assay was carried out according tomanufacturer's instruction. Results of this assay confirmed that IL-1Hy1 partially inhibited IL-1β stimulated PGE₂ production in adose-dependent manner (about 40-60% inhibition at 1000-fold excess). Itis possible that more highly purified preparations of IL-1 Hy1 may showcomplete inhibition in this assay. In comparison, IL-1ra completely PGE₂inhibited production at 100-fold excess.

7.15 Example 15 Inhibition of IL-1β Induced IL-6 Production

Inhibition of Interleukin-1β induced IL-6 production was studied usinghuman endothelial cells from umbilical vein (Huvec). Huvec cells wereseeded at 2×10⁴ cells per well in a 96-well plate the day before cellstimulation. On the day of stimulation, cells were washed once withfresh medium (F12 medium with 100 μg/ml heparin, 50 μg/ml endothelialgrowth supplement and 10% fetal bovine serum) and replated with 200 μlof fresh medium [without supplements] in each well. The Huvec cells werethen stimulated with 100 pg/ml (final volume) of IL-1β. Although thisassay was done with IL-1β, any cytokine of interest can be used. To testIL-6 inhibition, different concentrations of IL-1Hy1 (ranging from 10×to 1000× the concentration of IL-1β) or IL-1ra (ranging from 10× to1000× IL-1β concentration) were added to the wells with the IL-1β.

After 16 hours of cell stimulation, the culture plate was spun for fiveminutes at 4000 rpm to remove cell debris. 100 μl of the supernatant wasremoved to test for the presence of IL-6 using a human IL-6 immunoassaykit (R&D Systems) according to the manufacturer's instructions.

IL-1Hy1 partially inhibited IL-1β-stimulated IL-6 production in adose-dependent manner. In view of the fact that IL-6 blocks productionof tumor necrosis factor (TNF), a pro-inflammatory cytokine, the factthat IL-1Hy1 only partially inhibits of IL-6 production by IL1-Hy1 maybe beneficial in the treatment of inflammatory disease states withIL1-Hy1 due to reduced side effects. It is possible that more highlypurified preparations of IL-1Hy1 may show complete inhibition in thisassay.

7.16 Example 16 Determination of Molecular Mass of IL-1Hy1 Expressed inHuman Cells

To determine the molecular mass of IL-1Hy1 expressed in human cells, thehuman cell line THP-1 was used in experiments designed to detect thepresence of IL-1Hy1. As described in Mulero, et al., BBRC 263(3):702-706(1999), THP-1 cells express moderate levels of IL-1Hy1 whendifferentiated with phorbol ester (PMA) and subsequently stimulated withlipopolysaccharide (LPS). Briefly, THP-1 cells (ATCC) were grown toconfluency under conditions and media described in Mulero et al., supra,and then treated with 200 ng/ml of PMA (Sigma) and incubated at 37° C.for 48 hours. The cells were then subjected to 2 μg/ml of LPS (Sigma)and incubated at 37° C. for 24 hours. The cells and media were thenharvested in the presence of protease inhibitors (pepstatin, leupeptinand aprotinin; Sigma). After harvesting, the cells were lysed in a PLBlysis buffer (Promega). The media was processed by centrifugation toremove cell debris and concentrated twenty fold using centriprep(Amicon).

Recombinant IL-1Hy1 was also produced as follows. The plasmid pYZ5829(T7 promoter) was constructed by digesting IL-1Hy1 cDNA with NdeI andBamHI and cloning the resulting fragment in a Ndel-BamHI digested pRSETB vector (Invitrogen). This construct contains the 5′ most methioninecodon of SEQ ID NO: 4. E. Coli BL21plysE (Invitrogen) cells weretransformed with this vector and cultured in 2L flasks (for yields ofabout 2 g wet cell paste per liter of culture). The cells were disruptedin 10 ml per g wet cell paste 20 mM sodium phosphate, pH 7.5, using anEmulsiflex homogenizer. 5M acetic acid was added to titrate the pH to4.8, thus precipitating E. coli proteins, and the cell suspension wascentrifuged. The IL-1Hy1 remains in the supernatant. 5M acetic acid wasadded to the supernatant to titrate to pH 4.5, precipitating additionalproteins, and the resulting suspension was centrifuged again. Theresulting supernatant was then loaded onto an SP-Sepharose cationexchange column (Pharmacia) at pH 4.5 The IL-1Hy1 was eluted with a 0 to250 mM NaCl linear gradient (10×column volume), giving >90% pureprotein. The protein-containing fractions were pooled, titrated to pH8.0 with 5M NaOH, and 1 M Tris was added to 50 mM. Ammonium sulfate wasadded to 0.75M and the pool was loaded onto a butyl-Sepharose column(Pharmacia). IL-1Hy1 was eluted with a reverse salt gradient (20 columnvolumes). The protein was concentrated to 5-10 mg/ml and desalted bybuffer exchange into 20 mM sodium phosphate buffer at pH 7 on SephadexG-25 (Pharmacia). The resulting solution was loaded onto a Q-Sepharose(Pharmacia) anion exchange column, and IL-1Hy1 was collected in the flowthrough. NaCl was added to 0.1M, the protein solution was concentratedto 25 mg/ml and stored at −70° C.

The THP-1 and the bacterially produced recombinant IL-1Hy1 proteinpreparations described above were loaded on an SDS-PAGE gel andtransferred to a filter (Immobilon-P, Millipore) by Western blotting.The filter was then probed with polyclonal anti-IL-1Hy1 antibodyspecific for IL-1Hy1 prepared by immunizing rabbits with IL-1Hy1peptide: RLTQLPENGGWNA using conventional methods [see, e.g., Harlow etal., “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratories,Cold Spring Harbor, N.Y. (1998)] and control preimmune serum from theimmunized rabbits.

No IL-1Hy1 was detected in the media alone. A 17 kDa band was detectedin the lane loaded with recombinant IL-1Hy1 protein prepared from E.coli (translated from the 5′ most initiating methionine codon). Twobands were detected in the THP-1 cell lysate preparation: a faint bandthat corresponded to the same molecular mass as the recombinantbacterially expressed IL-1Hy1, representing a molecular mass ofapproximately 17 kDa; and a predominant band representing a molecularmass of approximately 15.5 kDa. The presence of the approximately 15.5kDa band suggested that the naturally occurring IL-1Hy1 protein may beprocessed in mammalian cells. In addition, the mature putative processedform is significantly smaller than the 17 kDa form expressed from theinitiating methionine codon commencing at nucleotide position 73 of SEQID NO: 4 (corresponding to amino acid position 1 of SEQ ID NO: 5). It ispossible that the ˜15.5 kDa IL-1Hy1 is the product of an alternativestart methionine codon commencing at nucleotide position 103 of SEQ IDNO: 4 (corresponding to amino acid position 11 of SEQ ID NO: 5), whichwould give a predicted molecular mass of about 16 kDa. It is alsopossible that the ˜15.5 kDa IL-1Hy1 is the product of proteolyticprocessing after the aspartic acid at amino acid position 13 of SEQ IDNO: 5.

SEQ ID NO: 5 is the deduced amino acid sequence corresponding to an openreading frame identified in SEQ ID NO: 4. The invention contemplatespolypeptide fragments of SEQ ID NO: 5 (either C-terminal or N-terminalfragments) having a molecular mass as determined by SDS-PAGE of about 16kDa or less, preferably about 15.5 kDa. The invention also specificallycontemplates N-terminally truncated fragments of SEQ ID NO: 4 thatcommence at either amino acid residue 11 or 14, as well aspolynucleotides encoding such N-terminally truncated fragments.

Example 17 IL-1Hy1 Expression in Activated THP-1 Cells

THP-1 cells were activated with PMA and LPS exactly as described abovein Example 16; cells and media were harvested after an additional 24hours in serum-free media, and cell lysates were prepared as describedabove. Control, non-activated THP-1 cells were also prepared byculturing cells for an equal amount of time in media without PMA or LPS,and cell lysates were prepared as described above.

Western blotting detected IL-1Hy1 protein in the cell lysate fromactivated THP-1 cells, while only a very little amount of IL-1Hy1 wasdetected in the cell lysate from non-activated cells. These resultsindicate that IL-1Hy1 protein expression is upregulated in activatedTHP-1 cells.

Example 18 Detection of IL-1Hy1 Protein Expression in Human Tissues byImmunohistochemistry

Slides of three different human tissue samples (tonsil, skin, andallergic nasal polyps from patients suffering from chronic allergyconditions) were stained with the rabbit polyclonal anti-IL1Hy1 antibodyprepared described above in Example 16. Anti-IL-1Hy1 antibody bindingwas detected by biotinylated goat-anti-rabbit secondary antibodyfollowed by streptavidin-HRP detection. To visually detect staining, theslides were treated with the chromogen 3,3′-diaminobenzidine (DAB; abrown stain) and counter stained with hematoxylin (blue nuclear stain).A negative control was stained in the same way in the absence ofanti-IL-1 Hy1 antibody.

In addition, double label staining was done as follows (see Myers, J.A., Mehta, P., Hunter, A. W., Berstein, S. A., and Erickson, P. A.,“Automated Double-Label: Immunohistochemistry”, Journal of SurgicalPathology, 1:105-113 (1995). Myers, J. A., D'Andrea, M. R., Hunter, A.W., Mehta, P., Berstein, S. A., and Erickson, P. A., “AutomatedDouble-Label: In Situ Hybridization and Immunohistochemistry”, Journalof Surgical Pathology, 1:191-203 (1995)) The anti-IL1Hy1 primaryantibody straining was done as described immediately above except thatfast red was used as the chromogen (a red stain). A second primaryantibody described below.(specific for cell phenotype markers) wasdetected using a biotinylated secondary antibody followed bystreptavidin-HRP. DAB was used as the chromogen (brown). The slides werecounter stained with hematoxylin (a blue nuclear stain) and werevisualized on a light microscope.

Results showed that the IL-1Hy1 protein was located in the cytoplasm ofskin epithelial cells. In the tonsil, there was positive staining inscattered cells in some germinal centers and epithelial crypts. Therewas no staining in T-cells (CD45RO marker), B cells (CD20 marker),macrophages (CD68 marker), or in monocytes, macrophages and Langerhanscells (CD14 marker).

Sections of the allergic nasal polyps were double label stained withantibodies against the IL-1Hy1 protein and IgE, and were double labelstained with anti-IL-1Hy1 and CD138. Tissue sections were then reactedwith secondary antibodies and streptavidin-HRP, and treated with thechromogen 3-amino-9-ethylcarbazole (AEC, red stain). In the allergicnasal polyps, IL-1Hy1 protein is expressed in plasma cells (CD138positive) that are IgE negative. These results suggest that IL-1 Hy1plays a role in modulating allergic reactions in the allergic nasalpolyps. Therefore, IL-1 Hy1 or antagonists of its activity (e.g.antibodies) may be useful in the treatment of allergic reactions, suchas allergic rhinitis and asthma. Effects of IL-1 Hy1 or antagoniststhereof can be confirmed in any of the allergy animal models describedherein or known in the art.

Serial section immunostaining was also performed on human tissues,including normal nasal tissue, chronic infected nasal polyps, allergicnasal polyps, normal lungs, and lung tissue from patients with chronicbronchitis due to chronic infection. Tissue sections were reacted withrabbit polyclonal anti-IL1-Hy1 antibody prepared as described in Example16 above. Anti-IL-1 Hy1 antibody binding was detected by biotinylatedgoat-anti-rabbit secondary antibody followed by streptavidin-APdetection. To visually detect staining, the slides were treated with thechromagen, (Fast Red) and counter stained with hematoxylin (blue nuclearstain).

The results demonstrate that allergic nasal polyps and chronicallyinfected nasal polyps had many more cells expressing L-1 Hy1 proteinthan the normal nasal polyps. Furthermore, the majority of the IL-1 Hy1expressing cells are IgA-producing plasma cells. In the lung tissuestested, chronic bronchitis lung tissues had many more IL-1 Hy1expressing cells than normal lung tissue. The IL-1 Hy1 expressing cellsincluded plasma cells, macrophages and bronchial epithelium cells;expression was highest in the plasma cells. These results suggest thatIL-1 Hy1 expressing cells are recruited to the site of allergic,infected or inflamed tissue and play a role in modulating inflammationdue to allergy and/or acute or chronic infection.

Example 19 Detection of IL-1Hy1 Protein Expression in Human Tissues byin-situ Hybridization Using DNA Probe

To determine tissue and cell types that express IL-1Hy1 mRNA, a 985nucleotide EcoRI and HindIII fragment (which included the complete IL-1Hy1 open reading frame) of the RTA00000273.c.07 clone from a fetalliver-spleen cDNA library was used as a probe on a panel of sectionedhuman tissues. The probe was labeled using the digoxigenin labeling kitsupplied by Boehringer-Mannheim using manufacturer's directions.Automated in-situ hybridization was performed by QualTek Molecular Labs(see Myers, et al., J. Surg. Path., 1:191-203 (1995). All tissues werefixed in 10% neutral buffered formalin, paraffin-embedded and cut into 4μm sections.

Cells in skin, brain and tonsil specifically hybridized to the IL-1Hy1probe. A strong signal was detected in the basal layer of the skinepithelia. Sporadic cells in the tonsil also produced a signal withstrong intensity. Brain cerebellum tissue provides evidence ofexpression in presumed infiltrating leukocytes. found surrounding anartery in the white matter. A different section of cerebellum from thesame individual exhibits staining of presumed glial cells located in themolecular layer.

Example 20 Detection of IL-1Hy1 mRNA Expression in Human Tissues byin-situ Hybridization Using Riboprobe

The following three pairs of riboprobes were labeled using thedigoxigenin labeling kit from Boehringer-Mannheim followingmanufacturer's directions.

Hy1-RNA1-5 5′-CACAGCTCCCGCCAGGAGAA-3′ Hy1-RNA1-35′-GGGACCACGCTGATCTCTTC-3′ Hy1-RNA2-5 5′-AGCTTCCCGAGAATGGTGGC-3′Hy1-RNA2-3 5′-GTGGTCAGGTGCCCACTAAG-3′ Hy1-RNA3-55′-CTGGGTAAGGAACTTAAAGAAC-3′ Hy1-RNA3-3 5′-TCTTAACTAACTACATCTGCA-3′

Serial sections of human normal tonsil were exposed to the DIG-labeledIL-1 Hy1 riboprobes and to antibodies for the following cell phenotypemarker proteins: CD20 (B cells), K167 (proliferating cells) CD3 (Tcells), CD1a (dendritic and langerhans cells), CD14 (monocytes,macrophages and langerhans cells), CD68 (macrophages), LN5 (macrophages,mantle zone cells and histocytes) and epithelial membrane antigen.Staining was done as described above.

The IL-1 Hy1 gene was expressed in scattered cells in some germinalcenters (in the area where B cells are activated) and in the epithelialcrypts. It was not clear whether it is expressed in macrophages or asubset of activated B cells.

Example 21 Induction of IL-13 by IL-1Hy1

The ability of IL-1 Hy1 to affect production of IL-13 was examined asfollows. One day prior to the experiment, normal human bronchialepithelial cells (NHBE) (Clonetics, San Diego, Calif.) or normal humansmall airway epithelial cells (SAEC) (Clonetics) were seeded at 8×10⁴cell/ml in one ml growth media (Clonetics). On the day of theexperiment, the cells were stimulated with either Il-1 Hy1 (1 μg/mlfinal concentration) or IL-1β (1 ng/ml final concentration). After 20hours incubation at 37° C. in 5% CO₂, cell supernatants were collected.Cell debris was removed by centrifugation and the amount of IL-13 in thesupernatant was quantitated by using an IL-13 ELISA kit (BiosourceInternational) according to the manufacturer's suggested protocol.

Results indicated that IL1-Hy1 was able to induce IL-13 production inboth cell types tested while IL-1β induced IL-13 production only in theNHBE cells at the concentration tested. In SAEC cells, IL-1 Hy1 inducedIL-13 production 110-fold over background, and in NHBE cells, IL-1 Hy1induced IL-13 production 60-fold. In NHBE cells, IL-1β induced IL-13production by the same 60-fold over background. These results, taken incombination with previous results from IL-6 and PGE₂ assays describedabove, suggests that an inhibitory effect of IL-1 Hy1 on IL-1β activitymay be indirect in that an increase in IL-13 production in turndecreases IL-1β synthesis.

Example 22 Inhibition of IL-18 Activity by IL-1 Hy1

The following experiment evaluated the ability of IL-1 Hy1 to inhibitIL-18 activity, as measured by induction of IFN-γ. Human lymphocytes(PBMC) were obtained by Ficoll-Hypaque density gradient separation ofperipheral blood from healthy volunteer donors. Immediately afterisolation, the PBMC were washed two times with growth media, containingRPMI 1640-10% fetal bovine serum., and 3×10⁵ cells/well were seeded in a96 well plate. The cells were stimulated by adding anti-CD3 antibody® &D Systems, Minneapolis, Minn.) to all of the samples at a finalconcentration of 0.5 μg/ml. At the time of stimulation, the wells werealso treated with a 100 ng/ml human recombinant IL-18 (R&D Systems) for36 hours at 37° C. at 5% CO₂. A portion of the wells on each plate(triplicates) were untreated to served as a measure of background levelsof IFN produced by stimulated PBMC cells. IL-18 treatment causes thePBMC cells to increase production of IFN-γ relative to the backgroundlevels.

To assay for IL-1 Hy1 inhibition of IL-18 stimulated IFNγ production,100× to 1000× fold concentration of IL-1 Hy1 (relative to IL-18concentration) was added to wells together with IL-18 at the time ofstimulation. After 36 hours of cell stimulation, the culture plate wascentrifuged for 5 minutes at 4000 rpm to remove cell debris. Thesupernatant was assayed for IFNγ using the Qantikine IFNγ ELISA kit® & DSystems) according to the manufacturer's suggested protocol.

Results indicated that IL-18 alone stimulated IFNγ production and thatIL-1 Hy1 inhibited the IL-18 stimulation in a dose dependent fashion,with complete inhibition observed at 500-fold to 1000-fold excess IL-1Hy1. In order to assess the mechanism by which IL-1 Hy1 reduced IFNγproduction, the following assay was carried out.

Human lymphocytes (PBMC) were obtained, washed, seeded, stimulated withanti-CD3 and treated with a final concentration of 100 ng/ml IL-18® & DSystems) as described above. Several blocking antibodies were then usedto test inhibition of IFNγ production, including anti-IL-18 receptorantibody, anti-Receptor accessory protein polyclonal antisera, anti-IL1receptor type I antibody and anti-IL1 receptor type II antibody (allobtained from R & D Systems, Minneapolis, Minn.). Different amounts ofeach antibody were added to the wells with IL-18, and after 36 hours ofcell stimulation, the culture plate was centrifuged for 5 min at 4000rpm to remove cell debris. The supernatant was assayed for IFNγ usingthe Qantikine IFNγ ELISA kit® & D Systems) according to manufacturer'sinstructions.

In the absence of an antibody, IL-18 stimulated IFNγ production relativeto background levels as observed above. However, anti-IL18 receptorantibody, anti-accessory protein antibody and anti-IL-1 receptor type I,but not type II, antibody inhibited IL-18 induced IFNγ production.

These results indicate that compounds which antagonize the action of theIL-1 receptor inhibit IL-18 activity as measured by induction of IFNγproduction.

Example 22 Binding of IL-1 Hy1 to the Interleukin-1 Receptor

A cell binding assay was carried out to determine if IL-1 Hy1 of theinvention binds to the interleukin-1 (IL-1) receptor. Briefly,fluorescent activated cell sorting (FACS) was used to measure cellbinding of the recombinant protein (see Example 12) in the presence andabsence of a 100-fold greater amount of unlabeled IL-1 receptorantagonist (IL-1 RA) ligand using fluorescent antibodies specific forthe express tag in the IL-1 Hy1 recombinant protein. In each reaction,10⁶ cells normal human dermal fibroblasts (NHDF ) were suspended in 100μl of FACS buffer (containing distilled PBS, 3% calf serum and 0.01%azide). Cell binding reactions included 5 nM recombinant IL-1 Hy1 in 100μl cell suspension; binding competition in one reaction included 500 nMof recombinant IL-1 RA. The cells were incubated on ice for one hr. Thecells were pelleted by centrifugation, 200 μl of 0.2 mM BS3 (crossliner)was added, and the cells were kept on ice for 30 min. Next, 10 μl 1 MTris pH 7.5 was added and the cells were incubated for 15 minutes onice. The cells were pelleted by centrifugation, washed one time in FACSbuffer, resuspended in 100 μl volume of FACS buffer, 2 μl primaryantibody (anti-express tag antibody 1 mg/ml) was added, and incubationcontinued on ice for an additional 30 min. The cells were pelleted bycentrifugation, washed with FACS buffer, and resuspended in FACS buffer(100 μl volume). The secondary antibody (phycoerythrin-conjugated), 2 μlof anti-mouse Ig (1 mg/ml), was added and the cells were incubated for30 minutes on ice. The cells were again pelleted by centrifugation,washed two times with FACS buffer, resuspended in 0.5 ml FACS buffer andanalyzed on FACS.

A shift in the fluorescence was observed for the cells treated with therecombinant tagged IL-1 Hy1. This binding was specific, as binding wasdecreased in the presence of the non-tagged IL-1 RA protein. Theseresults indicate binding of the IL-1 Hy1 protein of the invention to theIL-1 receptor.

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention, and compositions and methods which are functionallyequivalent are within the scope of the invention. Indeed, numerousmodifications and variations in the practice of the invention areexpected to occur to those skilled in the art upon consideration of thepresent preferred embodiments. Consequently, the only limitations whichshould be placed upon the scope of the invention are those which appearin the appended claims. All references cited within the body of theinstant specification are hereby incorporated by reference in theirentirety.

30 1 357 DNA Homo sapiens misc_feature (1)...(357) n = A,T,C or G 1ccgactctaa cactagagcc agtgaacatc atggagctct atcttggtgc caaggaatcc 60aagagcttca ccttctaccg gcgggacatg gggctcacct ccagcttcga gtcggctgcc 120tacccgggct ggttcctgtg cacggtgcct gaagccgatc agcctgtcag actcacccag 180cttcccgaga atggtggctg gaatgccccc atcacagact tctacttcca gcagtgtgac 240tagggcaacg tgccccccag aactccctgg gcagagccag ctcggntgan gggngagngn 300nnnnnnnnnn ngnnnnnnnn nnnnnnnnnn nnnnnnnnna nnnnnnnnnn nnnnnng 357 2 985DNA Homo sapiens CDS (1)...(240) 2 ccg act cta aca cta gag cca gtg aacatc atg gag ctc tat ctt ggt 48 Pro Thr Leu Thr Leu Glu Pro Val Asn IleMet Glu Leu Tyr Leu Gly 1 5 10 15 gcc aag gaa tcc aag agc ttc acc ttctac cgg cgg gac atg ggg ctc 96 Ala Lys Glu Ser Lys Ser Phe Thr Phe TyrArg Arg Asp Met Gly Leu 20 25 30 acc tcc agc ttc gag tcg gct gcc tac ccgggc tgg ttc ctg tgc acg 144 Thr Ser Ser Phe Glu Ser Ala Ala Tyr Pro GlyTrp Phe Leu Cys Thr 35 40 45 gtg cct gaa gcc gat cag cct gtc aga ctc acccag ctt ccc gag aat 192 Val Pro Glu Ala Asp Gln Pro Val Arg Leu Thr GlnLeu Pro Glu Asn 50 55 60 ggt ggc tgg aat gcc ccc atc aca gac ttc tac ttccag cag tgt gac 240 Gly Gly Trp Asn Ala Pro Ile Thr Asp Phe Tyr Phe GlnGln Cys Asp 65 70 75 80 tagggcaacg tgccccccag aactccctgg gcagagccagctcgggtgag gggtgagtgg 300 aggagaccca tggcggacaa tcactctctc tgctctcaggacccccacgt ctgacttagt 360 gggcacctga ccactttgtc ttctggttcc cagtttggataaattctgag atttggagct 420 cagtccacgg tcctccccca ctggatggtg ctactgctgtggaaccttgt aaaaaccatg 480 tggggtaaac tgggaataac atgaaaagat ttctgtgggggtggggtggg ggagtggtgg 540 gaatcattcc tgcttaatgg taactgacaa gtgttaccctgagccccgca ggccaaccca 600 tccccagttg agccttatag ggtcagtagc tctccacatgaagtcctgtc actcaccact 660 gtgcaggaga gggaggtggt catagagtca gggatctatggcccttggcc cagccccacc 720 cccttccctt taatcctgcc actgtcatat gctacctttcctatctcttc cctcatcatc 780 ttgttgtggg catgaggagg tggtgatgtc agaagaaatggctcgagctc agaagataaa 840 agataagtag ggtatgctga tcctctttta aaaacccaagatacaatcaa aatcccagat 900 gctggtctct attcccatga aaaagtgctc atgacatattgagaagacct acttacaaag 960 tggcatatat ttgcaattaa tttta 985 3 80 PRT Homosapiens 3 Pro Thr Leu Thr Leu Glu Pro Val Asn Ile Met Glu Leu Tyr LeuGly 1 5 10 15 Ala Lys Glu Ser Lys Ser Phe Thr Phe Tyr Arg Arg Asp MetGly Leu 20 25 30 Thr Ser Ser Phe Glu Ser Ala Ala Tyr Pro Gly Trp Phe LeuCys Thr 35 40 45 Val Pro Glu Ala Asp Gln Pro Val Arg Leu Thr Gln Leu ProGlu Asn 50 55 60 Gly Gly Trp Asn Ala Pro Ile Thr Asp Phe Tyr Phe Gln GlnCys Asp 65 70 75 80 4 1282 DNA Homo sapiens CDS (73)...(537) 4ccacgcgtcc gcacagctcc cgccaggaga aaggaacatt ctgaggggag tctacaccct 60gtggagctca ag atg gtc ctg agt ggg gcg ctg tgc ttc cga atg aag gac 111Met Val Leu Ser Gly Ala Leu Cys Phe Arg Met Lys Asp 1 5 10 tcg gca ttgaag gtg ctt tat ctg cat aat aac cag ctt cta gct gga 159 Ser Ala Leu LysVal Leu Tyr Leu His Asn Asn Gln Leu Leu Ala Gly 15 20 25 ggg ctg cat gcaggg aag gtc att aaa ggt gaa gag atc agc gtg gtc 207 Gly Leu His Ala GlyLys Val Ile Lys Gly Glu Glu Ile Ser Val Val 30 35 40 45 ccc aat cgg tggctg gat gcc agc ctg tcc ccc gtc atc ctg ggt gtc 255 Pro Asn Arg Trp LeuAsp Ala Ser Leu Ser Pro Val Ile Leu Gly Val 50 55 60 cag ggt gga agc cagtgc ctg tca tgt ggg gtg ggg cag gag ccg act 303 Gln Gly Gly Ser Gln CysLeu Ser Cys Gly Val Gly Gln Glu Pro Thr 65 70 75 cta aca cta gag cca gtgaac atc atg gag ctc tat ctt ggt gcc aag 351 Leu Thr Leu Glu Pro Val AsnIle Met Glu Leu Tyr Leu Gly Ala Lys 80 85 90 gaa tcc aag agc ttc acc ttctac cgg cgg gac atg ggg ctc acc tcc 399 Glu Ser Lys Ser Phe Thr Phe TyrArg Arg Asp Met Gly Leu Thr Ser 95 100 105 agc ttc gag tcg gct gcc tacccg ggc tgg ttc ctg tgc acg gtg cct 447 Ser Phe Glu Ser Ala Ala Tyr ProGly Trp Phe Leu Cys Thr Val Pro 110 115 120 125 gaa gcc gat cag cct gtcaga ctc acc cag ctt ccc gag aat ggt ggc 495 Glu Ala Asp Gln Pro Val ArgLeu Thr Gln Leu Pro Glu Asn Gly Gly 130 135 140 tgg aat gcc ccc atc acagac ttc tac ttc cag cag tgt gac 537 Trp Asn Ala Pro Ile Thr Asp Phe TyrPhe Gln Gln Cys Asp 145 150 155 tagggcaacg tgccccccag aactccctgggcagagccag ctcgggtgag gggtgagtgg 597 aggagaccca tggcggacaa tcactctctctgctctcagg acccccacgt ctgacttagt 657 gggcacctga ccactttgtc ttctggttcccagtttggat aaattctgag atttggagct 717 cagtccacgg tcctccccca ctggatggtgctactgctgt ggaaccttgt aaaaaccatg 777 tggggtaaac tgggaataac atgaaaagatttctgtgggg gtggggtggg ggagtggtgg 837 gaatcattcc tgcttaatgg taactgacaagtgttaccct gagccccgca ggccaaccca 897 tccccagttg agccttatag ggtcagtagctctccacatg aagtcctgtc actcaccact 957 gtgcaggaga gggaggtggt catagagtcagggatctatg gcccttggcc cagccccacc 1017 cccttccctt taatcctgcc actgtcatatgctacctttc ctatctcttc cctcatcatc 1077 ttgttgtggg catgaggagg tggtgatgtcagaagaaatg gctcgagctc agaagataaa 1137 agataagtag ggtatgctga tcctcttttaaaaacccaag atacaatcaa aatcccagat 1197 gctggtctct attcccatga aaaagtgctcatgacatatt gagaagacct acttacaaag 1257 tggcatatat ttgcaattaa tttta 1282 5155 PRT Homo sapiens 5 Met Val Leu Ser Gly Ala Leu Cys Phe Arg Met LysAsp Ser Ala Leu 1 5 10 15 Lys Val Leu Tyr Leu His Asn Asn Gln Leu LeuAla Gly Gly Leu His 20 25 30 Ala Gly Lys Val Ile Lys Gly Glu Glu Ile SerVal Val Pro Asn Arg 35 40 45 Trp Leu Asp Ala Ser Leu Ser Pro Val Ile LeuGly Val Gln Gly Gly 50 55 60 Ser Gln Cys Leu Ser Cys Gly Val Gly Gln GluPro Thr Leu Thr Leu 65 70 75 80 Glu Pro Val Asn Ile Met Glu Leu Tyr LeuGly Ala Lys Glu Ser Lys 85 90 95 Ser Phe Thr Phe Tyr Arg Arg Asp Met GlyLeu Thr Ser Ser Phe Glu 100 105 110 Ser Ala Ala Tyr Pro Gly Trp Phe LeuCys Thr Val Pro Glu Ala Asp 115 120 125 Gln Pro Val Arg Leu Thr Gln LeuPro Glu Asn Gly Gly Trp Asn Ala 130 135 140 Pro Ile Thr Asp Phe Tyr PheGln Gln Cys Asp 145 150 155 6 2648 DNA Homo sapiens 6 cacagctcccgccaggagaa aggaacattc tgaggggagt ctacaccctg tggagctcaa 60 gatggtcctgagtggggcgc tgtgcttccg aatgaaggac tcggcattga aggtgcttta 120 tctgcataataaccagcttc tagctggagg gctgcatgca gggaaggtca ttaaaggtga 180 agagatcagcgtggtcccca atcggtggct ggatgccagc ctgtcccccg tcatcctggg 240 tgtccagggtggaagccagt gcctgtcatg tggggtgggg caggagccga ctctaacact 300 agagccagtgaacatcatgg agctctatct tggtgccaag gaatccaaga gcttcacctt 360 ctaccggcgggacatggggc tcacctccag cttcgagtcg gctgcctacc cgggctggtt 420 cctgtgcacggtgcctgaag ccgatcagcc tgtcagactc acccagcttc ccgagaatgg 480 tggctggaatgcccccatca cagacttcta cttccagcag tgtgactagg gcaacgtgcc 540 ccccagaactccctgggcag agccagctcg ggtgaggggt gagtggagga gacccatggc 600 ggacaatcactctctctgct ctcaggaccc ccacgtctga cttagtgggc acctgaccac 660 tttgtcttctggttcccagt ttggataaat tctgagattt ggagctcagt ccacggtcct 720 cccccactggatggtgctac tgctgtggaa ccttgtaaaa accatgtggg gtaaactggg 780 aataacatgaaaagatttct gtgggggtgg ggtgggggag tggtgggaat cattcctgct 840 taatggtaactgacaagtgt taccctgagc cccgcaggcc aacccatccc cagttgagcc 900 ttatagggtcagtagctctc cacatgaagt cctgtcactc accactgtgc aggagaggga 960 ggtggtcatagagtcaggga tctatggccc ttggcccagc cccaccccct tccctttaat 1020 cctgccactgtcatatgcta cctttcctat ctcttccctc atcatcttgt tgtgggcatg 1080 aggaggtggtgatgtcagaa gaaatggctc gagctcagaa gataaaagat aagtagggta 1140 tgctgatcctcttttaaaaa cccaagatac aatcaaaatc ccagatgctg gtctctattc 1200 ccatgaaaaagtgctcatga catattgaga agacctactt acaaagtggc atatatttgc 1260 aattaattttaattaaaaga tacctattta tatatttctt tatagaaaaa agtctggaag 1320 agtttacttcaattgtagca atgtcagggt ggtggcagta taggtgattt ttcttttaat 1380 tctgttaatttatctgtatt tcctaatttt tctacaatga agatgaattc cttgtataaa 1440 aataagaaaagaaattaatc ttgaggtaag cagagcagac atcatctctg attgtcctca 1500 gcctccacttccccagagta aattcaaatt gaatcgagct ctgctgctct ggttggttgt 1560 agtagtgatcaggaaacaga tctcagcaaa gccactgagg aggaggctgt gctgaagttg 1620 tgtggctggaatctctgggt aaggaactta aagaacaaaa atcatctggt aattctttcc 1680 tagaaggatcacagcccctg ggattccaag gcattggatc cagtctctaa gaaggctgct 1740 gtactggttgaattgtgtcc ccctcaaatt cacatccttc ttggaatctc agtctgtgag 1800 tttatttggagataaggtct ctgcagatgt agttagttaa gacaaggtca tgctggatga 1860 aggtagacctaaattcaata tgactggttt ccttgtatga aaaggagagg acacagagac 1920 agaggagacgcggggaagac tatgtaaaga tgaaggcaga gatcggagtt ttgcagccac 1980 aagctaagaaacaccaagga ttgtggcaac catcagaagc ttggaagagg caaagaagaa 2040 ttcttccctagaggctttag agggataacg gctctgctga aaccttaatc tcagacttcc 2100 agcctcctgaacgaagaaag aataaatttc ggctgtttta agccaccaag gataattggt 2160 tacagcagctctaggaaact aatacagctg ctaaaatgat ccctgtctcc tcgtgtttac 2220 attctgtgtgtgtcccctcc cacaatgtac caaagttgtc tttgtgacca atagaatatg 2280 gcagaagtgatggcatgcca cttccaagat taggttataa aagacactgc agcttctact 2340 tgagccctctctctctgcca cccaccgccc ccaatctatc ttggctcact cgctctgggg 2400 gaagctagctgccatgctat gagcaggcct ataaagagac ttacgtggta aaaaatgaag 2460 tctcctgcccacagccacat tagtgaacct agaagcagag actctgtgag ataatcgatg 2520 tttgttgttttaaagttgct cagttttggt ctaacttgtt atgcagcaat agataaataa 2580 tatgcagagaaagagaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2640 aaaaaaaa2648 7 5751 DNA Homo sapiens misc_feature (1)...(5751) n = A,T,C or G 7cngttaatcc taccactatg agnatgtctt atcatttagt angaagattt ttgctttttg 60cacatgaaaa ataggttgga aaaagtatag gttttgtgat ctgtgtatga aagctgtcta 120tagtacatgt gtatgtgtgg gagaaaaagt gttgtcattg gttttctrat gantcactma 180gaaaagcaag tattcacatt ttttcttgtg gctgtctgat tttcaggttt ttctacaatg 240acatgtaggc tgancattcc ctargcagag agtcccacyt ctaacatctc ytgtaggcct 300ggcaakgcag cagaaasnca gaggaaggaa ggaggragaa ggaaggagtg aagraaggag 360taaaaaggta aggaagaaag ggaatagggr agraagggag raaatgggaa gggaaaraag 420gaaaggaagg aaagagggag ggaagaaagr aagggaaaag ggagggagtg agtgaatgaa 480agatggaaag aagraagaaa gggagggagg cagggaggaa agaaagttgc gcttcccttg 540asctgccatg ggcactgmyt cttagggtct gaaagcccct gagatgcaaa agcctagtgc 600tcacaaagag ctggaaagcc tcaaggaagt tcttcaatat ttctggaagg aaactgtctc 660cagaagcttc cctccccacg acagataatg agcagcaagt gcttctggcg acttagggtg 720atgtgaaatc acgctgggaa tcctgctcct cctcaggtcc tggcagtttc agggcccctc 780cctaggcctt acttaaaagg ctgaggcatc cttggaggaa caggcagact ccacagctcc 840cgccaggaga aaggaacatt ctgaggtatg ctctggggcg ctggtggtac cggagctctc 900tcctgacccc agacccagaa tctgctccgt ggaggctgtt cacatgctgg ggagctcggt 960gcagctgctt gctccccaga ccccagccaa ctcagcctct ctctccatga ttttctgttg 1020tttattccaa aataggggag tctacaccct gtggagctca agatggtcct gagtggggcg 1080ctgtgcttcc ggtgagtgta tgaggccctg gtttggtggt gtcctccgga ggaagtgagt 1140tctggataga cccgttgtcc agctctgagc aggagggagg aagggagggg ctgccattgs 1200rgctgkbaaa ttgtgaccag cacctcattg ctcttagagt tttcccagcc tttttcaaat 1260aggggcagga ctggggcarg ccatctcaca aggggwccct gatgctgagg gggacaagtg 1320aacctcccag wctaragctc cagccaagtc tatccaaggt gggaacgggg gccaggatcc 1380ctgctcagag ctccgccatt gtcccccatc acagtgaatg gatgtaagct cacccactct 1440gtgcccctac ctycctgcta ctctttgggg ataatwataa aacaaaaacc attaccatca 1500gccagtytgt mcacccactg gcatgtacca agccagacac tctgccgtgt tctgggctta 1560acaacagagg atgaaragtg ggcctttctc tcagkctaat aaagvacttc ccacgatgkg 1620ttctatggga ctcgattaga ggagkcccca gaggcatcca ggagatgctt tacacagkgg 1680agctctctga tcaagtaaaw gcagggaatw ctgctttcta catcctctca taagagaacc 1740acagcccagc tcagcatatg agwgactgag gktttctgaa gkaaggcaac ttgttgaatc 1800gyattdagct atgcatcgac ccaattttta cactgcatcc ttttccccca tataactttt 1860ggagaaaccc actttaggat acatcttcca cctcatagga tgccaggaaa tcaactgagt 1920tcaaagatga gaaacaactt tgaaaagtta aataaaagaa atttaaattt aaagaamctc 1980ctcacttagt aaggaatata tgaccaaata gaaatmcatg tatcttgaag aattgaagaa 2040tcaggcttta acgtggaaga ggcctggatg twatccmccc catcatctta gtgtagcaat 2100ggggaggctm aracccagag tgggcgagag agttgtctcc tgcgactcag cagcttggag 2160gmatagatgg ggcaagatcc tagggctstg actcaccgsc agcttctctt ccaacagtag 2220atgggttggg acagaaaagg ttaaataggg tsaagsakct wrccmcasay tccagtgkga 2280gactgtgrgg tcatcctcct tgtagrgcat gakcccagca gggctgrgag acaargctgt 2340gctgttactt ctggctacag tagkaagaaa gagagacaaa atgcytgagw ycmgggggyy 2400cyctggatcc agggcakgct gragtgtcca ccctcctcct aatgtagtcc tcwccccttc 2460ctgatgtttc agaatgaagg actcggcatt gaaggtgctt tatctgcata ataaccagct 2520tctagctgga gggctgcatg cagggaaggt cattaaaggt yggtratgaa acatgaccca 2580ctttcckkgg kctctataca ctctcagggg agggggcctg aagagggctt agaatagtca 2640tacagattak cataggccta crgagcccag gcattagggc aghacaaacc aggctctaag 2700caaaggcaaa taaaatacta cacctmtcag caaagtgaag acacacgctc tggggccacc 2760tgaagcttyt gtgcagaagt gagaatgttt tccaakakgc ttgtcttgty attcccttac 2820aggtagatwt aggtcaagca ttgcattccc tgggagccag taagtaccaa ggagagaact 2880aacgtagatt ctctatacct tttttcccat atgggagwgg gtttctgcct ctccaccctg 2940ggtcccctct gctctctgaa gatcctcagt cacttagagt ggagggaccc agagaacagg 3000tggcattgwt ggacctcctg cttgctcact mtgcmccatg cactgcaaca ggtccctcts 3060taaaatagtt ygcacctgcc cacctggggc acccttgctg agcwcagatg ccaggtagat 3120ccktcagcta ggccatatgt gtatgygtgt gcttactggt gkatgdatgt gtgcatscag 3180gcatatatgt gtrarcatat gtgtscatgc atgtatctgt atgtaaccat gtatgtgtra 3240gtgcagktat gtaggtatga scatgtgtgt gaatatgtat atgtgtscat gcatgtatct 3300gtgcatgtat gatctgatgt atgtgggtgg tgagggratg tacagagagg aatgagaccc 3360tcttttgctc tcagcarcct cacagggtgt agaaagttgt ccaamcaatt ccaaaggggg 3420gcttattaag acagggttca gaaaaaggcc tgagacccaa ggggcattaa aggagggggt 3480tgagtctatt ttgggttgta gaggcttgaa gatttgmccc tgaaytagag ggtggagtgg 3540aggtggtaca atgtgcttcc atgccttgat gtccactctg ggccagtgga caggagaanc 3600catgtmatgc cagctgctra gaagcctccc ttctgcccag cctgggggca ggccgtctca 3660cagcagtcyt gtgccataga rsgcaggaca rggaraaaag aaggaaaggc atccaggccy 3720tgcatctggc ntttttccca caggtgaaga gatcaagcgt ggtccccaat cggtggctgg 3780atgccagcct gtcccccgtc atcttgggtg tccagggtgg aagccagtgc ctgtcatgtg 3840gggtggggca ggagccgact cttaacacta gaggtgagac ttggggcatc ctcactgggg 3900actcagccac agatgctgag cctactgaag ccgggcagyc cacagccytg gtgctgtggg 3960acaccctagc aggattctgt tgatggcagc tttgcctcct ccmtaaggat cctgcccagc 4020cctccctctg cccctgcttc tgccctcacc tgacctcccc tcctctgccg gcagccagtg 4080aacatcatgg agctctatct tggtgccaag gaatccaaga gcttcacctt ctaccggcgg 4140gacatggggc tcacctccag cttcgagtcg gctgcctacc cgggctggtt cctgtgcacg 4200gtgcctgaag ccgatcagcc tgtcagactc acccagcttc ccgagaatgg tggctggaat 4260gcccccatca cagacttcta cttccagcag tgtgactagg gcaacgtgcc ccccagaact 4320ccctgggcag agccagctcg ggtgaggggt gagtggagga gacccatggc ggacaatcac 4380tctctctgct ctcaggaccc ccacgtctga cttagtgggc acctgaccac tttgtcttct 4440ggttcccagt ttggataaat tctgagattt ggagctcagt ccacggtcct cccccactgg 4500atggtgctac tgctgtggaa ccttgtaaaa accatgtggg gtaaactggg aataacatga 4560aaagatttct gtgggggtgg ggtgggggag tggtgggaat cattcctgct taatggtaac 4620tgacaagtgt taccctgagc cccgcaggcc aacccatccc cagttgagcc ttatagggtc 4680agtagctctc cacatgaagt cctgtcactc accactgtgc aggagaggga ggtggtcata 4740gagtcaggga tctatggccc ttggcccagc cccaccccct tccctttaat cctgccactg 4800tcatatgcta cctttcctat ctcttccctc atcatcttgt tgtgggcatg aggaggtggt 4860gatgtcagaa gaaatggctc gagctcagaa gataaaagat aagtagggta tgctgatcct 4920cttttaaaaa cccaagatac aatcaaaatc cccagatgct ggtctctatt cccatgaaaa 4980agtgctcatg acatattgag aagacctact tacaaagtgg catatattgc aatttatttt 5040aattaaaaga tacctattta tatatttctt tatagaaaaa agtctggaag agtttacttc 5100aattgtagca atgtcagggt ggtggcagta taggtgatwt ttcttttaat tctgttaatt 5160tatctgtatt tcctaatttt tctacaatga agatgaattc cttgtataaa aataagaaaa 5220gaaattaatc ttgaggtaag cagagcagac atcatctctg atkgcctcag cctccacttc 5280cccagagtaa attcaaattg aatcgagctc tgctgctctg gttggttgta gtagtgatca 5340ggaawcagat ctcagcaaag ccactgagga ggaggctgtg atgagtttgt gtggctggaa 5400tctctgggta aggaacttaa agaacaaaaa tcatctggta attctttcct agaaggatca 5460cagcccctgg gattccaagg cattggatcc agtctctaag aaggctgctg tactggttga 5520attgtgtccc cctcaaattc acatccttct tggaatctca gtctgtgagt ttatttggag 5580ataaggtctc tgcagatgta gttagttaag acaaggtcat gctggatgaa ggtagaccta 5640aattyaatat gactggttty cttgtatgaa aaggagagga cacagagaca gaggagacgc 5700ggggaagact atgtaaagat gaaggcagag atcggagttt tgcagccaca a 5751 8 7605 DNAHomo sapiens 8 aatattgaca gtatgcacag tcatagtttc attttactta ttatttatttatttattgag 60 gcagaagtct agctctcttc tgtcatccag ctgggagtac agtggctcaatcttggctca 120 ccgcaatctc acctccaggt tcaagcaatt ctcacacctc agcctcctgagtagcttgga 180 ttacaaatgt gcactacaac ccggctaatt tttgtgtttt cagtaaagatggggttttgc 240 catgttggcc aggcttgtct catttcatct tatttctact gtcattccatttggtcaatt 300 tgtaatttaa taattttgta caaggcagtt tctacattaa ttttatttttctgaaaagtg 360 tctatcatat tagtggcatt atgaaaatcg tagattattt tctgtgttttgaacaacttg 420 atattttatt tattcttgaa cacttgtagg acctcttggc tcatatgtctgaataccatt 480 tctttaaaat gtacgcttta acatgtatac atatgtaaca aacctgcacattgtacacat 540 gtatcctaaa acttaaagta taataataat aataaaagaa aaaagaaaaataaaacttaa 600 aaaaataaag tgtatgcttt aacaataaat tattaataaa tagttatctgtgttattcaa 660 gatataatta aattgttttt tgtgcactga cccttacctt tatttcagatccatcacaga 720 gagtgcatgg aggtatttaa actgcattac tgtttcacaa ttaagtattatctttatcaa 780 ttaggattca agaaagatca cttacagaat tatagatggc atgagctagattttactttc 840 taaagaaata accagataca tgagcaaaga tgttaataca aagatgtttgtcacaacatg 900 gttttcaata gcaaaaaaag agagaaaaat atataaaaga caaataacagtggataggtt 960 tcaataaata atgttacagt gatacagtta aatactatac agctattgaagcatgtcatt 1020 attcatattt agtatggaaa gatattttgc tattttgcta catgaaaaaatgaggttgga 1080 aaaagtatag gttttgtgaa tctgttgtat gaaagctgtc ttatagttacatgtgtatgt 1140 gtgtggagga aaaagtgttg tcattggttt tctgatgatg cactcagaaaagacaagtat 1200 tcacattttt tcttgtggct gatctggatt ttcaggtttt tctacaatgaacatgtaggc 1260 tgaacattcc ctaagcagga gagtcccacc tctaacatct cctgtaggcctggcaatggc 1320 aggcaggaaa gacagaggaa ggaaggaggg agaagggaag gagtgaaggaaggagtgaaa 1380 aaggtaagga agaaagggaa taggggagga agggaggaaa tgggaagggaaagaaggaaa 1440 kgaagggaaa gagggagggg aagaaaggaa ggggaaaagg gagggagtgagttgaatgaa 1500 agatggaaag aaggaagaaa gggagggagg cagggaggaa agaaagttgcgcttcccttg 1560 agctgcccat gggcacctga ctcttagggt ctgaaaggcc cctgagatgcaaaagcctag 1620 tgctcacaaa gagctggaaa gcctcaagga agttcttcaa tatttctggaaggaaactgt 1680 ctccagaagc ttccctcccc acgacagata atgagcagca agtgcttctggcgacttagg 1740 gtgatgtgaa attcacgctg ggaatcctgc tcctcctcag gtcctggcaagtttcagggc 1800 ccctccctag gccttactta aaaggctgag gcatccttgg aggaacaggcagactccaca 1860 gctcccgcca ggagaaagga acattctgag gtatgctctg gggcgctggtggtaccggag 1920 ctctctcctg accccagacc cagaatctgc tccgtggagg ctgttcacatgctggggagc 1980 tcggtgcagc tgcttgctcc ccagacccca gccaactcag cctctctctccatgattttc 2040 tgttgtttat tccaaaatag gggagtctac accctgtgga gctcaagatggtcctgagtg 2100 gggcgctgtg cttccggtga gtgtatgagg ccctggtttg gtggtgtcctccggaggaag 2160 tgagttctgg atagacccgt tgtccagctc tgagcaggag ggaggaagggagggggctgc 2220 cattgcagct gggaaattgt gaccagcacc tcattgctct tagagttttcccagcctttt 2280 tcaaataggg gcaggactgg ggcaggccat ctcacaaggg gtccctgatgctgaggggga 2340 caagtgaacc tcccagtcta gagctccagc caagtctatc caaggtgggaacgggggcca 2400 ggatccctgc tcagagctcc gccattgtcc cccatcacag tgaatggatgtaagctcacc 2460 cactctgtgc ccctacctcc ctgctactct ttgggggata ataataaaacaaaaaccatt 2520 accatcagcc aagtctgtcc acccactggc atgtaccaag ccagacactctgccgtgttc 2580 tgggcttaac aaccagagga tgagagtggt cctttctctc agtctaataaagcacttccc 2640 acgatgtgtt ctatgggact cgattagagg agtcccacag aggcatccaggagatgcttt 2700 acacagtgga gctctctgat caagtaaatg cagggaattc tgctttctacatcctctcat 2760 aagagaacca cagcccagct cagcatatga gtgactgagg ktttctgaagtaaggcaact 2820 tgttgaatcg yatttagcta tgcatcgacc caatttttac actgcatccttttcccccat 2880 ataacttttg gagaaaccca ctttaggata catcttccac ctcataggatgccaggaaat 2940 caactgagtt caaagatgag aaacaacttt gaaaagttaa ataaaagaaatttaaattta 3000 aagaaactcc tcacttagta aggaatatat gaccaaatag aaatacatgtatcttgaaga 3060 attgaagaat caggctttaa cgtggaagag gcctggatgt tatccaacccatcatcttag 3120 tgtagcaatg gggaggctca gacccaagag tgggcgagag agttgtctcctgcgactcag 3180 cagcattgga ggcatagatg gggcaagatc ctagggctct gactcaccgagcagcttctc 3240 ttccaacagg agatgggttg gggcagaaaa ggttgaatag ggtgaaggagcaaaccacag 3300 actccagtgg gagactgtgg ggtcatcctc cttgtagggc atgagcccagcagggctggg 3360 agacaaggct gtgctgttac ttctggcaca gtaggaagaa agagagacaaaatgcctgag 3420 atcagggggt tctctggatc cagggcatgc tggagtgtcc accctcctcctaatgtagtc 3480 ctcacccctt cctgatgttt cagaatgaag gactcggcat tgaaggtgctttatctgcat 3540 aataaccagc ttctagctgg agggctgcat gcagggaagg tcattaaaggttggtgatga 3600 aacatgaccc actttccttg gtctctatac actctcaggg gagggggcctgaagagggct 3660 tagaatagtc atacagatta gcataggcct acagagccca ggcattagggcagcacaaac 3720 caggctctaa gcaaaggcaa ataaaatact acacctctca gcaaagtgaagacacacgct 3780 ctggggccac ctgaagcttc tgtgcagaag tgagaatgtt ttccaagaggcttgtcttgt 3840 cattccctta caggtagatw taggtcaagc attgcattcc ctgggagccagtaagtacca 3900 aggagagaac taacgtagat tctctatacc ttttttccca tatgggagtgggtttctgcc 3960 tctccaccct gggtcccctc tgctctctga agatcctcag tcacttagagtggagggacc 4020 cagagaacag gtggcattgt tggacctcct gcttgctcac tctgccccatgcactgcaac 4080 aggtccctct ctaaaatagt tygcacctgc ccacctgggg cacccttgctgagcacagat 4140 gccaggtaga tccttcagct aggccatatg tgtatgtgtg tgcttactggtgtatgtatg 4200 tgtgcatgca ggcatatatg tgtgagcata tgtgtgcatg catgtatctgtatgtaacca 4260 tgtatgtgtg agtgcaggta tgtaggtatg agcatgtgtg tgaatatgtatatgtgtgca 4320 tgcatgtatc tgtgcatgta tgatctgatg tatgtgggtg gtgaggggatgtacagagag 4380 gaatgagacc ctcttttgct ctcagcaacc tcacagggtg tagaaagttgtccaaacaat 4440 tccaaagggg ggcttattaa gacagggttc agaaaaaggc ctgagacccaaggggcatta 4500 aaggaggggg ttgagtctat tttgggttgt agaggcttga agatttgaccctgaactaga 4560 gggtggagtg gaggtggtac aatgtgcttc catgccttga tgtccactctgggccagtgg 4620 acaggagaag ccatgtcatg acagctgctg agaagcctcc cttctgcccagcctgggggc 4680 aggccgtctc acagcagtcc tgtgccctag agcccaggac aggggaagaaggagggaaag 4740 gcatccaggg ccctgcatct ggcctctttc ccacaggtga agagatcagcgtggtcccca 4800 atcggtggct ggatgccagc ctgtcccccg tcatcctggg tgtccagggtggaagccagt 4860 gcctgtcatg tggggtgggg caggagccga ctcttaacac tagaggtgagacttggggca 4920 tcctcactgg ggactcagcc acagatgctg agcctactga agccgggcagcccacagccc 4980 tggtgctgtg ggacacccta gcaggattct gttgatggca gctttgcctcctccctaagg 5040 atcctgccca gccctccctc tgcccctgct tctgccctca cctgacctcccctcctctgc 5100 cggcagccag tgaacatcat ggagctctat cttggtgcca aggaatccaagagcttcacc 5160 ttctaccggc gggacatggg gctcacctcc agcttcgagt cggctgcctacccgggctgg 5220 ttcctgtgca cggtgcctga agccgatcag cctgtcagac tcacccagcttcccgagaat 5280 ggtggctgga atgcccccat cacagacttc tacttccagc agtgtgactagggcaacgtg 5340 ccccccagaa ctccctgggc agagccagct cgggtgaggg gtgagtggaggagacccatg 5400 gcggacaatc actctctctg ctctcaggac ccccacgtct gacttagtgggcacctgacc 5460 actttgtctt ctggttccca gtttggataa attctgagat ttggagctcagtccacggtc 5520 ctcccccact ggatggtgct actgctgtgg aaccttgtaa aaaccatgtggggtaaactg 5580 ggaataacat gaaaagattt ctgtgggggt ggggtggggg agtggtgggaatcattcctg 5640 cttaatggta actgacaagt gttaccctga gccccgcagg ccaacccatccccagttgag 5700 ccttataggg tcagtagctc tccacatgaa gtcctgtcac tcaccactgtgcaggagagg 5760 gaggtggtca tagagtcagg gatctatggc ccttggccca gccccacccccttcccttta 5820 atcctgccac tgtcatatgc tacctttcct atctcttccc tcatcatcttgttgtgggca 5880 tgaggaggtg gtgatgtcag aagaaatggc tcgagctcag aagataaaagataagtaggg 5940 tatgctgatc ctcttttaaa aacccaagat acaatcaaaa tcccagatgctggtctctat 6000 tcccatgaaa aagtgctcat gacatattga gaagacctac ttacaaagtggcatatattg 6060 caatttattt taattaaaag atacctattt atatatttct ttatagaaaaaagtctggaa 6120 gagtttactt caattgtagc aatgtcaggg tggtggcagt ataggtgatttttcttttaa 6180 ttctgttaat ttatctgtat ttcctaattt ttctacaatg aagatgaattccttgtataa 6240 aaataagaaa agaaattaat cttgaggtaa gcagagcaga catcatctctgattgcctca 6300 gcctccactt ccccagagta aattcaaatt gaatcgagct ctgctgctctggttggttgt 6360 agtagtgatc aggaatcaga tctcagcaaa gccactgagg aggaggctgtgatgagtttg 6420 tgtggctgga atctctgggt aaggaactta aagaacaaaa atcatctggtaattctttcc 6480 tagaaggatc acagcccctg ggattccaag gcattggatc cagtctctaagaaggctgct 6540 gtactggttg aattgtgtcc ccctcaaatt cacatccttc ttggaatctcagtctgtgag 6600 tttatttgga gataaggtct ctgcagatgt agttagttaa gacaaggtcatgctggatga 6660 aggtagacct aaattcaata tgactggttt ccttgtatga aaaggagaggacacagagac 6720 agaggagacg cggggaagac tatgtaaaga tgaaggcaga gatcggagttttgcagccac 6780 aagctaagaa acaccaagga ttgtggcaac catcagaagc ttggaagaggcaaagaagaa 6840 ttcttcccta gaggctttag agggataacg gctctgctga caccttaatctcagacttcc 6900 agcctcctga acgaagaaag aataaatttc ggctgtttta agccaccaaggataattggt 6960 tatggcagct ctaggaaact aatacagctg ctaaaatgat ccctgtctcctcgtgtttac 7020 attctgtgtg tgtcccctcc cacaatgtac caaagttgtc tttgtgaccaatagaatatg 7080 gcagaagtga tggcatgcca cttccaagat taggttataa aagacactgcagcttctact 7140 tgagccctct ctctctgcca cccaccgccc ccaatctatc ttggctcactcgctctgggg 7200 gaagctagct tccatgctat gagcaggcct ataaagagac ttatgtggtaaaaaatgaag 7260 tctcctgccc acagccacat tagtgaacct agaagcagag actctgtgagataatcaatg 7320 tttgttgttt taagttgctc agttttggtc taacttgtta tgcagcaatagataaataat 7380 atgcagagaa agagaaacaa atgcatttgt tttattattg caattttctccaatattttt 7440 tattttcttt ctcacaatga acaactatcc ttcatttacc caaatattctatttaaaagc 7500 taataataca gcatttgttg agtcatctgg ttctgcaaga ttgagatcctcttgtcctat 7560 gtgccaggaa tgaactccag tgccccaccc aaaccctggg gaatg 7605 9178 PRT Mus musculus 9 Met Glu Ile Cys Trp Gly Pro Tyr Ser His Leu IleSer Leu Leu Leu 1 5 10 15 Ile Leu Leu Phe His Ser Glu Ala Ala Cys ArgPro Ser Gly Lys Arg 20 25 30 Pro Cys Lys Met Gln Ala Phe Arg Ile Trp AspThr Asn Gln Lys Thr 35 40 45 Phe Tyr Leu Arg Asn Asn Gln Leu Ile Ala GlyTyr Leu Gln Gly Pro 50 55 60 Asn Ile Lys Leu Glu Glu Lys Ile Asp Met ValPro Ile Asp Leu His 65 70 75 80 Ser Val Phe Leu Gly Ile His Gly Gly LysLeu Cys Leu Ser Cys Ala 85 90 95 Lys Ser Gly Asp Asp Ile Lys Leu Gln LeuGlu Glu Val Asn Ile Thr 100 105 110 Asp Leu Ser Lys Asn Lys Glu Glu AspLys Arg Phe Thr Phe Ile Arg 115 120 125 Ser Glu Lys Gly Pro Thr Thr SerPhe Glu Ser Ala Ala Cys Pro Gly 130 135 140 Trp Phe Leu Cys Thr Thr LeuGlu Ala Asp Arg Pro Val Ser Leu Thr 145 150 155 160 Asn Thr Pro Glu GluPro Leu Ile Val Thr Lys Phe Tyr Phe Gln Glu 165 170 175 Asp Gln 10 178PRT Rattus norvegicus 10 Met Glu Ile Cys Arg Gly Pro Tyr Ser His Leu IleSer Leu Leu Leu 1 5 10 15 Ile Leu Leu Phe Arg Ser Glu Ser Ala Gly HisPro Ala Gly Lys Arg 20 25 30 Pro Cys Lys Met Gln Ala Phe Arg Ile Trp AspThr Asn Gln Lys Thr 35 40 45 Phe Tyr Leu Arg Asn Asn Gln Leu Ile Ala GlyTyr Leu Gln Gly Pro 50 55 60 Asn Thr Lys Leu Glu Glu Lys Ile Asp Met ValPro Ile Asp Phe Arg 65 70 75 80 Asn Val Phe Leu Gly Ile His Gly Gly LysLeu Cys Leu Ser Cys Val 85 90 95 Lys Ser Gly Asp Asp Thr Lys Leu Gln LeuGlu Glu Val Asn Ile Thr 100 105 110 Asp Leu Asn Lys Asn Lys Glu Glu AspLys Arg Phe Thr Phe Ile Arg 115 120 125 Ser Glu Thr Gly Pro Thr Thr SerPhe Glu Ser Leu Ala Cys Pro Gly 130 135 140 Trp Phe Leu Cys Thr Thr LeuGlu Ala Asp His Pro Val Ser Leu Thr 145 150 155 160 Asn Thr Pro Lys GluPro Cys Thr Val Thr Lys Phe Tyr Phe Gln Glu 165 170 175 Asp Gln 11 177PRT Oryctolagus cuniculus 11 Met Arg Pro Ser Arg Ser Thr Arg Arg His LeuIle Ser Leu Leu Leu 1 5 10 15 Phe Leu Phe His Ser Glu Thr Ala Cys ArgPro Ser Gly Lys Arg Pro 20 25 30 Cys Arg Met Gln Ala Phe Arg Ile Trp AspVal Asn Gln Lys Thr Phe 35 40 45 Tyr Leu Arg Asn Asn Gln Leu Val Ala GlyTyr Leu Gln Gly Pro Asn 50 55 60 Ala Lys Leu Glu Glu Arg Ile Asp Val ValPro Leu Glu Pro Gln Leu 65 70 75 80 Leu Phe Leu Gly Ile Gln Arg Gly LysLeu Cys Leu Ser Cys Val Lys 85 90 95 Ser Gly Asp Lys Met Lys Leu His LeuGlu Ala Val Asn Ile Thr Asp 100 105 110 Leu Gly Lys Asn Lys Glu Gln AspLys Arg Phe Thr Phe Ile Arg Ser 115 120 125 Asn Ser Gly Pro Thr Thr ThrPhe Glu Ser Ala Ser Cys Pro Gly Trp 130 135 140 Phe Leu Cys Thr Ala LeuGlu Ala Asp Gln Pro Val Ser Leu Thr Asn 145 150 155 160 Thr Pro Asp AspSer Ile Val Val Thr Lys Phe Tyr Phe Gln Glu Asp 165 170 175 Gln 12 18PRT Homo sapiens 12 Asn Tyr Pro Lys Lys Lys Met Glu Lys Arg Phe Val PheAsn Lys Ile 1 5 10 15 Glu Ile 13 18 PRT Homo sapiens 13 Leu Ser Glu AsnArg Lys Gln Asp Lys Arg Phe Ala Phe Ile Arg Ser 1 5 10 15 Asp Ser 14 159PRT Homo sapiens 14 Met Ala Leu Glu Thr Ile Cys Arg Pro Ser Gly Arg LysSer Ser Lys 1 5 10 15 Met Gln Ala Phe Arg Ile Trp Asp Val Asn Gln LysThr Phe Tyr Leu 20 25 30 Arg Asn Asn Gln Leu Val Ala Gly Tyr Leu Gln GlyPro Asn Val Asn 35 40 45 Leu Glu Glu Lys Ile Asp Val Val Pro Ile Glu ProHis Ala Leu Phe 50 55 60 Leu Gly Ile His Gly Gly Lys Met Cys Leu Ser CysVal Lys Ser Gly 65 70 75 80 Asp Glu Thr Arg Leu Gln Leu Glu Ala Val AsnIle Thr Asp Leu Ser 85 90 95 Glu Asn Arg Lys Gln Asp Lys Arg Phe Ala PheIle Arg Ser Asp Ser 100 105 110 Gly Pro Thr Thr Ser Phe Glu Ser Ala AlaCys Pro Gly Trp Phe Leu 115 120 125 Cys Thr Ala Met Glu Ala Asp Gln ProVal Ser Leu Thr Asn Met Pro 130 135 140 Asp Glu Gly Val Met Val Thr LysPhe Tyr Phe Gln Glu Asp Glu 145 150 155 15 21 DNA Gene-specific 5′primer 15 ccccactgga tggtgctact g 21 16 21 DNA Gene-specific 3′ primer16 gggaagagat aggaaaggta g 21 17 12 DNA Intron 17 tctgaggtat gc 12 18 12DNA Exon 18 aaatagggga gt 12 19 12 DNA Intron 19 cttccggtga gt 12 20 12DNA Exon 20 tttcagaatg aa 12 21 12 DNA Intron 21 ttaaaggttg gt 12 22 12DNA Exon 22 ccacaggtga ag 12 23 12 DNA Intron 23 ctagaggtga ga 12 24 12DNA Exon 24 cggcagccag tg 12 25 29 DNA Artificial Sequence TBD 25gaagatctat ggtcctgagt ggggccctg 29 26 29 DNA Artificial Sequence TBD 26gaagatctgt cacactgctg gaagtagaa 29 27 12 DNA Artificial Sequence TBD 27ctgtaggcct gg 12 28 12 DNA Artificial Sequence TBD 28 aaaaaggtaa gg 1229 12 DNA Artificial Sequence TBD 29 cctcaggtcc tg 12 30 177 PRT Homosapiens 30 Met Glu Ile Cys Arg Gly Leu Arg Ser His Leu Ile Thr Leu LeuLeu 1 5 10 15 Phe Leu Phe His Ser Glu Thr Ile Cys Arg Pro Ser Gly ArgLys Ser 20 25 30 Ser Lys Met Gln Ala Phe Arg Ile Trp Asp Val Asn Gln LysThr Phe 35 40 45 Tyr Leu Arg Asn Asn Gln Leu Val Ala Gly Tyr Leu Gln GlyPro Asn 50 55 60 Val Asn Leu Glu Glu Lys Ile Asp Val Val Pro Ile Glu ProHis Ala 65 70 75 80 Leu Phe Leu Gly Ile His Gly Gly Lys Met Cys Leu SerCys Val Lys 85 90 95 Ser Gly Asp Glu Thr Arg Leu Gln Leu Glu Ala Val AsnIle Thr Asp 100 105 110 Leu Ser Glu Asn Arg Lys Gln Asp Lys Arg Phe AlaPhe Ile Arg Ser 115 120 125 Asp Ser Gly Pro Thr Thr Ser Phe Glu Ser AlaAla Cys Pro Gly Trp 130 135 140 Phe Leu Cys Thr Ala Met Glu Ala Asp GlnPro Val Ser Leu Thr Asn 145 150 155 160 Met Pro Asp Glu Gly Val Met ValThr Lys Phe Tyr Phe Gln Glu Asp 165 170 175 Glu

What is claimed is:
 1. An isolated polynucleotide probe consistingessentially of a fragment of SEQ ID NO: 4 which specifically hybridizesto the nucleotide sequence of SEQ ID NO: 4 or its complement under thefollowing hybridization conditions: washing at 68° C. in a solutioncontaining 0.1×SSC and 0.1% SDS.
 2. A polynucleotide probe of claim 1,wherein the fragment is the nucleotide sequence of SEQ ID NO:
 2. 3. Apolynucleotide probe of claim 1 which is bound to an array.